Anti-PSMA Antibodies, Bispecific Antigen-Binding Molecules That Bind PSMA and CD3, and Uses Thereof

ABSTRACT

The present invention provides antibodies that bind to prostate-specific membrane antigen (PSMA), bispecific antibodies that bind to PSMA and CD3, and methods of using the same. According to certain embodiments, the antibodies of the invention bind human PSMA with high affinity and bind CD3 to induce human T cell proliferation. The invention includes antibodies that bind PSMA and CD3 and induce T cell-mediated killing of PSMA-expressing tumor cells. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3, and a second antigen-binding molecule that specifically binds human PSMA. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of prostate tumors expressing PSMA. The antibodies and bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced targeted immune response is desired and/or therapeutically beneficial. For example, the antibodies of the invention are useful for the treatment of various cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/205,917,filed Nov. 30, 2018, which is a division of U.S. application Ser. No.15/223,434, filed Jul. 29, 2016, which claims the benefit under 35 USC §119(e) of US Provisional Application Nos. 61/199,823, filed Jul. 31,2015, 62/222,590, filed Sep. 23, 2015, and 62/351,823, filed Jun. 17,2016, each of which is herein incorporated by reference.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 10173US03-Sequence.txt,created on Sep. 16, 2021 and containing 630,228 bytes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for prostate-specific membraneantigen (PSMA), and methods of use thereof. The present invention alsorelates to bispecific antigen-binding molecules that bind PSMA and CD3,and methods of use thereof.

BACKGROUND

Prostate-specific membrane antigen (PSMA), also known as folatehydrolase 1 (FOLH1), is an integral, non-shed membrane glycoprotein thatis highly expressed in prostate epithelial cells and is a cell-surfacemarker for prostate cancer. Its expression is maintained incastrate-resistant prostate cancer, a condition with poor outcome andlimited treatment options. Methods for treating prostate cancer bytargeting PSMA have been investigated. For example, Yttrium-90 capromabis a radiotherapeutic comprising a monoclonal antibody to anintracellular epitope of PSMA. In another example, J591, a monoclonalantibody to an extracellular epitope of PSMA, is part of theradiotherapeutic Lutetium-177 J591 and in MLN2704, in which maytansinoid1 (DM1, an antimicrotubule agent) is conjugated to J591. These therapieshave been associated with toxicity. PSMA is also expressed within theneovasculature of other tumors such as bladder, renal, gastric, andcolorectal carcinomas.

CD3 is a homodimeric or heterodimeric antigen expressed on T cells inassociation with the T cell receptor complex (TCR) and is required for Tcell activation. Functional CD3 is formed from the dimeric associationof two of four different chains: epsilon, zeta, delta and gamma. The CD3dimeric arrangements include gamma/epsilon, delta/epsilon and zeta/zeta.Antibodies against CD3 have been shown to cluster CD3 on T cells,thereby causing T cell activation in a manner similar to the engagementof the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodieshave been proposed for therapeutic purposes involving the activation ofT cells. In addition, bispecific antibodies that are capable of bindingCD3 and a target antigen have been proposed for therapeutic usesinvolving targeting T cell immune responses to tissues and cellsexpressing the target antigen.

Antigen-binding molecules that target PSMA, as well as bispecificantigen-binding molecules that bind both PSMA and CD3 would be useful intherapeutic settings in which specific targeting and T cell-mediatedkilling of cells that express PSMA is desired.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides antibodies andantigen-binding fragments thereof that bind to human PSMA. Theantibodies according to this aspect of the invention are useful, interalia, for targeting cells expressing PSMA. The present invention alsoprovides bispecific antibodies and antigen-binding fragments thereofthat bind human PSMA and human CD3. The bispecific antibodies accordingto this aspect of the invention are useful, inter alia, for targeting Tcells expressing CD3, and for stimulating T cell activation, e.g., undercircumstances where T cell-mediated killing of cells expressing PSMA isbeneficial or desirable. For example, the bispecific antibodies candirect CD3-mediated T cell activation to specific PSMA-expressing cells,such as prostate tumor cells.

Exemplary anti-PSMA antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs) and light chainvariable regions (LCVRs), as well as heavy chain complementaritydetermining regions (HCDR1, HCDR2 and HCDR3), and light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) of theexemplary anti-PSMA antibodies. Table 2 sets forth the sequenceidentifiers of the nucleic acid molecules encoding the HCVRs, LCVRs,HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-PSMAantibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-PSMA antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is selected from the group consistingof SEQ ID NOs: 66/1642 (e.g., H1H11810P2); and 122/130 (e.g., H1H3465P).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-PSMA antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is selected fromthe group consisting of SEQ ID NOs: 72/1648 (e.g., H1H11810P2) and128/136 (e.g., H1H3465P).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-PSMA antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of SEQ ID NOs:68-70-72-1644-1646-1648 (e.g., H1H11810P2); and 124-126-128-132-134-136(e.g., H1H3465P).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary anti-PSMAantibodies listed in Table 1. For example, the present inventionincludes antibodies, or antigen-binding fragments thereof, comprisingthe HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences setcontained within an HCVR/LCVR amino acid sequence pair selected from thegroup consisting of SEQ ID NOs: 66/146 (e.g., H1H11810P2); and 122/130(e.g., H1H3465P). Methods and techniques for identifying CDRs withinHCVR and LCVR amino acid sequences are well known in the art and can beused to identify CDRs within the specified HCVR and/or LCVR amino acidsequences disclosed herein. Exemplary conventions that can be used toidentify the boundaries of CDRs include, e.g., the Kabat definition, theChothia definition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-PSMA antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs HCDR1-HCDR2-HCDR3),wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined byany of the exemplary anti-PSMA antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs LCDR1-LCDR2-LCDR3),wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined byany of the exemplary anti-PSMA antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-PSMA antibody listed in Table1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-PSMA antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-PSMA antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds PSMA and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-PSMA antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-PSMA antibody. Additionalcombination therapies and co-formulations involving the anti-PSMAantibodies of the present invention are disclosed elsewhere herein.

In another aspect, the invention provides therapeutic methods fortargeting/killing tumor cells expressing PSMA using an anti-PSMAantibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an anti-PSMA antibody of the invention to asubject in need thereof. In some cases, the anti-PSMA antibodies (orantigen-binding fragments thereof) can be used for treating prostatecancer, or may be modified to be more cytotoxic by methods, includingbut not limited to, modified Fc domains to increase ADCC (see e.g.Shield et al. (2002) JBC 277:26733), radioimmunotherapy (Akhtar, et al.,2012, Prostate-Specific Membrane Antigen-Based Therapeutics; Adv Urol.2012: 973820), antibody-drug conjugates (Olson, W C and Israel, R J,2014, Front Biosci (Landmark Ed). 19:12-33; DiPippo, et al. Feb. 15,2015, The Prostate, 75(3):303-313, first published on line Oct. 18,2014), or other methods for increasing the efficiency of tumor ablation.

The present invention also includes the use of an anti-PSMA antibody ofthe invention in the manufacture of a medicament for the treatment of adisease or disorder related to or caused by PSMA-expressing cells.

In yet another aspect, the invention provides monospecific anti-PSMAantibodies for diagnostic applications, such as, e.g., imaging reagents.

In yet another aspect, the invention provides therapeutic methods forstimulating T cell activation using an anti-CD3 antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody

In another aspect, the present invention provides an isolated antibodyor antigen-binding fragment thereof that binds human prostate-specificmembrane antigen (PSMA) with a binding dissociation equilibrium constant(K_(D)) of less than about 80 nM as measured in a surface plasmonresonance assay at 37° C. In yet another aspect, the present inventionprovides an isolated antibody or antigen-binding fragment thereof thatbinds human PSMA with a dissociative half-life (t½) of greater thanabout 10 minutes as measured in a surface plasmon resonance assay at 37°C.

The invention further provides an antibody or antigen-binding fragmentthat competes for binding to human PSMA with a reference antibodycomprising an HCVR/LCVR amino acid sequence pair as set forth inTable 1. In another aspect, the invention provides an antibody orantigen-binding fragment that competes for binding to human PSMA with areference antibody comprising an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 2/1642; 10/1642;18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642;82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.

The invention furthermore provides an antibody or antigen-bindingfragment, wherein the antibody or antigen-binding fragment thereof bindsto the same epitope on human PSMA as a reference antibody comprising anHCVR/LCVR amino acid sequence pair as set forth in Table 1. In anotheraspect, the antibody or antigen-binding fragment binds to the sameepitope on human PSMA as a reference antibody comprising an HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs:2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642;58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642;114/1642; 122/130; and 138/146.

The invention further provides an isolated antibody or antigen-bindingfragment thereof that binds human PSMA, wherein the antibody orantigen-binding fragment comprises: the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR) having an aminoacid sequence as set forth in Table 1; and the CDRs of a light chainvariable region (LCVR) having an amino acid sequence as set forth inTable 1. In another aspect, the isolated antibody or antigen-bindingfragment comprises the heavy and light chain CDRs of a HCVR/LCVR aminoacid sequence pair selected from the group consisting of: SEQ IDNOs:2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642;58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642;114/1642; 122/130; and 138/146. In yet another aspect, the isolatedantibody or antigen-binding fragment comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected fromthe group consisting of: SEQ ID NOs: 4-6-8-1644-1646-1648;12-14-16-1644-1646-1648; 20-22-24-1644-1646-1648;28-30-32-1644-1646-1648; 36-38-40-1644-1646-1648;44-46-48-1644-1646-1648; 52-54-56-1644-1646-1648;60-62-64-1644-1646-1648; 68-70-72-1644-1646-1648;76-78-80-1644-1646-1648; 84-86-88-1644-1646-1648;92-94-96-1644-1646-1648; 100-102-104-1644-1646-1648;108-110-112-1644-1646-1648; 116-118-120-1644-1646-1648;124-126-128-132-134-136; and 140-142-144-148-150-152.

In another aspect, the invention provides an isolated antibody orantigen-binding fragment thereof that binds human PSMA, wherein theantibody or antigen-binding fragment comprises: (a) a heavy chainvariable region (HCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74,82, 90, 98, 106, 114, 122, and 138; and (b) a light chain variableregion (LCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 130 and 146. In a further aspect, the isolatedantibody or antigen-binding fragment of claim 10, wherein the antibodyor antigen-binding fragment comprises a HCVR/LCVR amino acid sequencepair selected from the group consisting of: SEQ ID NOs:2/1642; 10/1642;18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642;82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.

According to another aspect, the present invention provides bispecificantigen-binding molecules (e.g., antibodies) that bind PSMA and CD3.Such bispecific antigen-binding molecules are also referred to herein as“anti-PSMA/anti-CD3 bispecific molecules,” “anti-CD3/anti-PSMAbispecific molecules,” or “PSMAxCD3 bsAbs.” The anti-PSMA portion of theanti-PSMA/anti-CD3 bispecific molecule is useful for targeting cells(e.g., tumor cells) that express PSMA (e.g., prostate tumors), and theanti-CD3 portion of the bispecific molecule is useful for activatingT-cells. The simultaneous binding of PSMA on a tumor cell and CD3 on aT-cell facilitates directed killing (cell lysis) of the targeted tumorcell by the activated T-cell. The anti-PSMA/anti-CD3 bispecificmolecules of the invention are therefore useful, inter alia, fortreating diseases and disorders related to or caused by PSMA-expressingtumors (e.g., prostate cancers).

The bispecific antigen-binding molecules according to this aspect of thepresent invention comprise a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds PSMA. The present invention includesanti-PSMA/anti-CD3 bispecific molecules (e.g., bispecific antibodies)wherein each antigen-binding domain comprises a heavy chain variableregion (HCVR) paired with a light chain variable region (LCVR). Incertain exemplary embodiments of the invention, the anti-CD3antigen-binding domain and the anti-PSMA antigen binding domain eachcomprise different, distinct HCVRs paired with a common LCVR. Forexample, as illustrated in Example 4 herein, bispecific antibodies wereconstructed comprising a first antigen-binding domain that specificallybinds CD3, wherein the first antigen-binding domain comprises anHCVR/LCVR pair derived from an anti-CD3 antibody; and a secondantigen-binding domain that specifically binds PSMA, wherein the secondantigen-binding domain comprises an HCVR derived from an anti-PSMAantibody paired with an LCVR derived from an anti-CD3 antibody (e.g.,the same LCVR that is included in the anti-CD3 antigen-binding domain).In other words, in the exemplary molecules disclosed herein, the pairingof an HCVR from an anti-PSMA antibody with an LCVR from an anti-CD3antibody creates an antigen-binding domain that specifically binds PSMA(but does not bind CD3). In such embodiments, the first and secondantigen-binding domains comprise distinct anti-CD3 and anti-PSMA HCVRsbut share a common anti-CD3 LCVR. In other embodiments, the bispecificantigen-binding molecules comprise distinct anti-CD3 and anti-PSMAHCVRs, but share a common LCVR. The amino acid sequence of this LCVR isshown, e.g., in SEQ ID NO:1386, and the amino acid sequences of thecorresponding CDRs LCDR1-LCDR2-LCDR3) are shown in SEQ ID NOs:1388, 1390and 1392, respectively. Genetically modified mice can be used to producefully human bispecific antigen-binding molecules comprising twodifferent heavy chains that associate with an identical light chain thatcomprises a variable domain derived from one of two different humanlight chain variable region gene segments. Alternatively, variable heavychains may be paired with one common light chain and expressedrecombinantly in host cells. As such, the antibodies of the inventioncan comprise immunoglobulin heavy chains associated with a singlerearranged light chain. In some embodiments, the light chain comprises avariable domain derived from a human VK1-39 gene segment or a VK3-20gene segment. In other embodiments, the light chain comprises a variabledomain derived from a human VK1-39 gene segment rearranged with a humanJK5 or a human JK1 gene segment.

The present invention provides anti-CD3/anti-PSMA bispecific molecules,wherein the first antigen-binding domain that specifically binds CD3comprises any of the HCVR amino acid sequences, any of the LCVR aminoacid sequences, any of the HCVR/LCVR amino acid sequence pairs, any ofthe heavy chain CDR1-CDR2-CDR3 amino acid sequences, or any of the lightchain CDR1-CDR2-CDR3 amino acid sequences as set forth in US publication2014/0088295.

In addition, the present invention provides anti-CD3/anti-PSMAbispecific molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises any of the HCVR amino acid sequences asset forth in Tables 12, 14, and 18 herein. The first antigen-bindingdomain that specifically binds CD3 may also comprise any of the LCVRamino acid sequences as set forth in Tables 12, 15, and 20 herein.According to certain embodiments, the first antigen-binding domain thatspecifically binds CD3 comprises any of the HCVR/LCVR amino acidsequence pairs as set forth in Tables 12, 14, 15, 18, and 20 herein. Thepresent invention also provides anti-CD3/anti-PSMA bispecific molecules,wherein the first antigen-binding domain that specifically binds CD3comprises any of the heavy chain CDR1-CDR2-CDR3 amino acid sequences asset forth in Tables 12, 14, and 18 herein, and/or any of the light chainCDR1-CDR2-CDR3 amino acid sequences as set forth in Tables 12, 15, and20 herein.

According to certain embodiments, the present invention providesanti-CD3/anti-PSMA bispecific molecules, wherein the firstantigen-binding domain that specifically binds CD3 comprises a heavychain variable region (HCVR) having an amino acid sequence as set forthin Tables 12, 14, and 18 herein or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a light chain variable region (LCVR) having an aminoacid sequence as set forth in Tables 12, 15, and 20 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequence pairas set forth in Tables 12, 14, 15, 18, and 20 herein.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence as set forth in Tables 12, 14, and 18 herein, or asubstantially similar sequence thereto having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR3 (LCDR3) domain having an amino acid sequence as set forth in Tables12, 15, and 20 herein, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In certain embodiments, the first antigen-binding domain thatspecifically binds CD3 comprises a HCDR3/LCDR3 amino acid sequence pairas set forth in Tables 12, 14, 15, 18, and 20 herein.

The present invention also provides anti-CD3/anti-PSMA bispecificantigen-binding molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises a heavy chain CDR1 (HCDR1) domainhaving an amino acid as set forth in Tables 12, 14, and 18 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2(HCDR2) domain having an amino acid as set forth in Tables 12, 14, and18, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a heavy chainCDR3 (HCDR3) domain having an amino acid as set forth in Tables 12, 14,and 18, or a substantially similar sequence thereof having at least 90%,at least 95%, at least 98% or at least 99% sequence identity; a lightchain CDR1 (LCDR1) domain having an amino acid sequence as set forth inTables 12, 15, and 20 herein, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a light chain CDR2 (LCDR2) domain having an aminoacid sequence as set forth in Tables 12, 15, and 20 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity, and a light chainCDR3 (LCDR3) domain having an amino acid sequence as set forth in Tables12, 15, and 20 herein, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Certain non-limiting, exemplary anti-CD3/anti-PSMA bispecificantigen-binding molecules of the invention include a firstantigen-binding domain that specifically binds CD3 comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences as set forth in Tables 12, 14, 15, 18, and 20herein.

The present invention further provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) comprising an amino acid sequence as set forth in Table 12, Table14, or Table 18 and light chain complementarity determining regions(LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR)comprising an amino acid sequence as set forth in Table 12, Table 15, orTable 20.

In another aspect, the invention provides a bispecific antigen-bindingmolecule wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) selected from the group consisting of SEQ ID NOs: 922, 154, 1482,1490, 1498, 1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578,1586, 1594, 1602, 1610, 1618, and 1626, and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) from a light chain variableregion (LCVR) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 162, 930 and 1642.

The invention further provides a bispecific antigen-binding molecule,wherein the first antigen-binding domain that specifically binds humanCD3 comprises three heavy chain complementarity determining regions(A1-HCDR1, A1-HCDR2 and Al-HCDR3) and three light chain complementaritydetermining regions (A1-LCDR1, Al-LCDR2 and A1-LCDR3), wherein A1-HCDR1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 924, 156; 1484, 1492, 1500, 1508, 1516, 1524, 1532, 1540,1548, 1556, 1564, 1572, 1580, 1588, 1596, 1604, 1612, 1620, and 1628;A1-HCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 926, 158, 1486, 1494, 1502, 1510, 1518, 1526,1534, 1542, 1550, 1558, 1566, 1574, 1582, 1590, 1598, 1606, 1614, 1622,and 1630; A1-HCDR3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 928, 160, 1488, 1496, 1504, 1512, 1520,1528, 1536, 1544, 1552, 1560, 1568, 1576, 1584, 1592, 1600, 1608, 1616,1624, and 1632; A1-LCDR1 comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 932, 164, and 1644; Al-LCDR2comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 166, 934 and 1646; and A1-LCDR3 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 168, 936 and1648.

In a further aspect, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises the heavy and light chain CDRs of a HCVR/LCVRamino acid sequence pair selected from the group consisting of: SEQ IDNOs: 922/930, 154/162, 1482/1642, 1490/1642, 1498/1642, 1506/1642,1514/1642, 1522/1642, 1530/1642, 1538/1642, 1546/1642, 1554/1642,1562/1642, 1570/1642, 1578/1642, 1586/1642, 1594/1642, 1602/1642,1610/1642, 1618/1642, and 1626/1642

In another aspect, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises three heavy chain complementarity determiningregions (Al-HCDR1, Al-HCDR2 and Al-HCDR3) and three light chaincomplementarity determining regions (A1-LCDR1, Al-LCDR2 and A1-LCDR3),and wherein the second antigen-binding domain that specifically bindshuman PSMA comprises three heavy chain complementarity determiningregions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chaincomplementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3);wherein A1-HCDR1 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 924, 156, 1484, 1492, 1500, 1508, 1516,1524, 1532, 1540, 1548, 1556, 1564, 1572, 1580, 1588, 1596, 1604, 1612,1620, and 1628; A1-HCDR2 comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 926, 158, 1486, 1494, 1502, 1510,1518, 1526, 1534, 1542, 1550, 1558, 1566, 1574, 1582, 1590, 1598, 1606,1614, 1622, and 1630; A1-HCDR3 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:928, 160, 1488, 1496, 1504,1512, 1520, 1528, 1536, 1544, 1552, 1560, 1568, 1576, 1584, 1592, 1600,1608, 1616, 1624, and 1632; A1-LCDR1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 164, 932, and 1644;A1-LCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 166, 934, and 1646; and A1-LCDR3 comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:168, 936, and 1648; and wherein A2-HCDR1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 124 and 68;A2-HCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 126 and 70; A2-HCDR3 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 128 and 72;A2-LCDR1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 932, 164, and 1644; A2-LCDR2 comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:934, 166, and 1646; and A2-LCDR3 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 936, 168, and 1648.

Certain non-limiting, exemplary anti-CD3/anti-PSMA bispecificantigen-binding molecules of the invention include a firstantigen-binding domain that specifically binds CD3 comprising a heavychain comprising variable domain framework regions having an amino acidsequence selected from FR1 (SEQ ID NO: 1654), FR2 (SEQ ID NO: 1656), FR3(SEQ ID NO: 1657), and FR4 (SEQ ID NO: 1658).

In more embodiments, exemplary anti-CD3/anti-PSMA bispecificantigen-binding molecules of the invention include a bispecificantigen-binding molecule wherein the first antigen-binding domain thatspecifically binds human CD3 comprises a HCVR comprisingHCDR1-HCDR2-HCDR3 having the amino acid sequences of SEQ ID NOs:1659-1660-1661.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the second antigen-binding domain that specificallybinds PSMA comprises a heavy chain variable region (HCVR) having theamino acid sequence selected from the group consisting of SEQ ID NOs:2,10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 138,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the second antigen-binding domain that specificallybinds PSMA comprises a light chain variable region (LCVR) having theamino acid sequence selected from the group consisting of SEQ IDNOs:930, 162, and 1642, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the second antigen-binding domain that specificallybinds PSMA comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequencepair selected from the group consisting of SEQ ID NOs: 122/930, 122/162,and 66/1642.

The present invention also provides anti-CD3/anti-PSMA bispecificmolecules, wherein the second antigen-binding domain that specificallybinds PSMA comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs:8, 16,24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, and 144, ora substantially similar sequence thereto having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR3 (LCDR3) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 936, 168, and 1648, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In certain embodiments, the second antigen-binding domain thatspecifically binds PSMA comprises a HCDR3/LCDR3 amino acid sequence pairselected from the group consisting of SEQ ID NOs: 128/936, 128/168, and72/1648.

The present invention also provides anti-CD3/anti-PSMA bispecificantigen-binding molecules, wherein the second antigen-binding domainthat specifically binds PSMA comprises a heavy chain CDR1 (HCDR1) domainhaving an amino acid sequence selected from the group consisting of SEQID NOs:4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116,124, and 140, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:6, 14, 22, 30, 38, 46, 54, 62,70, 78, 86, 94, 102, 110, 118, 126 and 142, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a heavy chain CDR3 (HCDR3) domain having anamino acid sequence selected from the group consisting of SEQ ID NOs: 8,16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, and 144,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a light chainCDR1 (LCDR1) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 932, 164, and 1644, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 934, 166, and 1646, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a light chain CDR3 (LCDR3) domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 936, 168, and1648, or a substantially similar sequence thereof having at least 90%,at least 95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary anti-CD3/anti-PSMA bispecificantigen-binding molecules of the invention include a secondantigen-binding domain that specifically binds PSMA comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences selected from the group consisting of: SEQ ID NOs:124-126-128-932-934-936, 124-126-128-164-166-168, and68-70-72-1644-1646-1648.

In a related embodiment, the invention includes anti-CD3/anti-PSMAbispecific antigen-binding molecules wherein the second antigen-bindingdomain that specifically binds PSMA comprises the heavy and light chainCDR domains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NOs:122/930, 122/162, and 66/1642.

In another aspect, the invention provides a bispecific antigen-bindingmolecule comprising a first antigen-binding domain that binds human CD3and a second antigen-binding domain that binds human PSMA, wherein thesecond antigen-binding domain is derived from the antibody orantigen-binding fragment of any one of the anti-PSMA antibodies of theinvention. In a further aspect, the invention provides a bispecificantigen-binding molecule comprising a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds human PSMA.

The invention further provides a bispecific antigen-binding moleculewhich binds human cells expressing human CD3 and cynomolgus monkey cellsexpressing cynomolgus CD3. In another aspect, the bispecificantigen-binding molecule binds human cells expressing human PSMA andcynomolgus monkey cells expressing cynomolgus PSMA.

In another aspect the invention provides a bispecific antigen-bindingmolecule which inhibits tumor growth in immunocompromised mice bearinghuman prostate cancer xenografts. The invention further provides abispecific antigen-binding molecule which inhibits tumor growth inimmunocompetent mice bearing human prostate cancer xenografts. Theinvention further provides a bispecific antigen-binding molecule whichsuppresses tumor growth of established tumors in immunocompromised micebearing human prostate cancer xenografts. The invention further providesa bispecific antigen-binding molecule which reduces tumor growth ofestablished tumors in immunocompetent mice bearing human prostate cancerxenografts.

In another aspect the invention provides a bispecific antigen-bindingmolecule comprising i) a first antigen-binding domain that specificallybinds an effector cell with an EC₅₀ value of greater than about 40 nMand, and ii) a second antigen-binding domain that specifically binds atarget human prostate tumor cell with an EC₅₀ value of less than 40 nM,wherein such EC₅₀ binding affinity value is measured in an in vitro FACSbinding assay.

For example, the bispecific antigen-binding molecule can include a firstantigen-binding domain that specifically binds human CD3 with an EC₅₀value of greater than about 40 nM, or greater than about 100 nM, greaterthan about 200 nM, or greater than about 1 μM. In one embodiment, thebispecific antigen-binding molecule can include a second antigen-bindingdomain that specifically binds the target prostate tumor cell with anEC₅₀ value of less than about 6 nM. In some cases, the firstantigen-binding domain specifically binds each of human CD3 andcynomolgus CD3 with an EC₅₀ value of greater than about 40 nM, greaterthan about 100 nM, greater than about 200 nM, or greater than about 1μM. In some cases, the first antigen-binding domain specifically bindseach of human CD3 and cynomolgus CD3 with weak or no measurableaffinity.

In some embodiments, the antigen-binding molecule induces Tcell-mediated tumor cell killing with an EC₅₀ value of less than about1.3 nM, as measured in an in vitro T cell-mediated tumor cell killingassay, for example, where the tumor cells are C4-2, 22Rv1, andTRAMPC2_PSMA cells.

In some applications, the first antigen-binding domain binds human CD3with an K_(D) value of greater than about 11 nM, as measured in an invitro surface plasmon resonance binding assay. In some instances, thefirst antigen-binding domain binds each of human CD3 and cynomolgus CD3with an K_(D) value of greater than about 15 nM, greater than about 30nM, greater than about 60 nM, greater than about 120 nM, or greater thanabout 300 nM, as measured in an in vitro surface plasmon resonancebinding assay.

In certain embodiments, anti-CD3 antibodies of the invention,antigen-binding fragments and bispecific antibodies thereof were made byreplacing amino acid residues of a parental in a stepwise manner basedon differences between the germline sequence and the parental antibodysequence.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the second antigen-binding domain competes for bindingto human PSMA with a reference antigen-binding protein comprising threeheavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 andA2-HCDR3) and three light chain complementarity determining regions(A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 124 and68; A2-HCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 126 and 70; A2-HCDR3 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 128 and 72;A2-LCDR1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 164, 932 and 1644; A2-LCDR2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 166, 934and 1646; and A2-LCDR3 comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs:168, 936, and 1648. In someembodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the second antigen-binding domain competes for bindingto human PSMA with a reference antigen-binding protein comprising aheavy chain variable region (HCVR) comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 122 and 66, and alight chain variable region (LCVR) comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 162, 930 and 1642.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain competes for bindingto human CD3 with a reference antigen-binding protein comprising threeheavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 andA1-HCDR3) and three light chain complementarity determining regions(A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 924,156, 1484, 1492, 1500, 1508, 1516, 1524, 1532, 1540, 1548, 1556, 1564,1572, 1580, 1588, 1596, 1604, 1612, 1620, and 1628; A1-HCDR2 comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:926, 158, 1486, 1494, 1502, 1510, 1518, 1526, 1534, 1542, 1550, 1558,1566, 1574, 1582, 1590, 1598, 1606, 1614, 1622, and 1630; A1-HCDR3comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 928, 160, 1488, 1496, 1504, 1512, 1520, 1528, 1536, 1544,1552, 1560, 1568, 1576, 1584, 1592, 1600, 1608, 1616, 1624, and 1632;A1-LCDR1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 164, 932, and 1644; A1-LCDR2 comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:166, 934, and 1646; and A1-LCDR3 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 168, 936, and 1648. Insome embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain competes for bindingto human CD3 with a reference antigen-binding protein comprising a heavychain variable region (HCVR) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 922, 154, 1482, 1490, 1498,1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578, 1586, 1594,1602, 1610, 1618, and 1626, and a light chain variable region (LCVR)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:930, 162, and 1642.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain competes for bindingto human CD3 with a reference antigen-binding protein comprising a heavychain variable region (HCVR) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 922, 154, 1482, 1490, 1498,1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578, 1586, 1594,1602, 1610, 1618, and 1626, and a light chain variable region (LCVR)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:930, 162, and 1642; and wherein the second antigen-bindingdomain competes for binding to human PSMA with a referenceantigen-binding protein comprising a heavy chain variable region (HCVR)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:122 and 66, and a light chain variable region (LCVR)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 930, 162, and 1642.

In one aspect, the invention provides a pharmaceutical compositioncomprising an anti-PSMA antigen-binding molecule or anti-PSMA/anti-CD3bispecific antigen-binding molecule and a pharmaceutically acceptablecarrier or diluent. The invention further provides a method for treatinga cancer in a subject, the method comprising administering to thesubject the pharmaceutical composition comprising an anti-PSMAantigen-binding molecule or anti-PSMA/anti-CD3 bispecificantigen-binding molecule and a pharmaceutically acceptable carrier ordiluent. In some embodiments, the cancer is selected from the groupconsisting of prostate cancer, kidney cancer, bladder cancer, colorectalcancer, and gastric cancer. In some cases, the cancer is prostatecancer. In some cases, the prostate cancer is castrate-resistantprostate cancer.

In another aspect, the present invention provides nucleic acid moleculesencoding any of the HCVR, LCVR or CDR sequences of theanti-CD3/anti-PSMA bispecific antigen-binding molecules disclosedherein, including nucleic acid molecules comprising the polynucleotidesequences as set forth in Tables 2, 13, 15, 17, 19, and 21 herein, aswell as nucleic acid molecules comprising two or more of thepolynucleotide sequences as set forth in Tables 2, 13, 15, 17, 19, and21 in any functional combination or arrangement thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

The present invention includes anti-CD3/anti-PSMA bispecificantigen-binding molecules wherein any of the aforementionedantigen-binding domains that specifically bind CD3 are combined,connected or otherwise associated with any of the aforementionedantigen-binding domains that specifically bind PSMA to form a bispecificantigen-binding molecule that binds CD3 and PSMA.

The present invention includes anti-CD3/anti-PSMA bispecificantigen-binding molecules having a modified glycosylation pattern. Insome applications, modification to remove undesirable glycosylationsites may be useful, or an antibody lacking a fucose moiety present onthe oligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, modification of galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising an anti-CD3/anti-PSMA bispecific antigen-binding molecule asdisclosed herein and a pharmaceutically acceptable carrier. In a relatedaspect, the invention features a composition which is a combination ofan anti-CD3/anti-PSMA bispecific antigen-binding molecule and a secondtherapeutic agent. In one embodiment, the second therapeutic agent isany agent that is advantageously combined with an anti-CD3/anti-PSMAbispecific antigen-binding molecule. Exemplary agents that may beadvantageously combined with an anti-CD3/anti-PSMA bispecificantigen-binding molecule are discussed in detail elsewhere herein.

In yet another aspect, the invention provides therapeutic methods fortargeting/killing tumor cells expressing PSMA using ananti-CD3/anti-PSMA bispecific antigen-binding molecule of the invention,wherein the therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ananti-CD3/anti-PSMA bispecific antigen-binding molecule of the inventionto a subject in need thereof.

The present invention also includes the use of an anti-CD3/anti-PSMAbispecific antigen-binding molecule of the invention in the manufactureof a medicament for the treatment of a disease or disorder related to orcaused by PSMA-expressing cells.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that PSMAxCD3 bispecific antibody inhibits growth ofa human prostate cell line in vivo. NSG mice were co-implanted with22Rv1 cells and human PBMCs subcutaneously. The animals were dosed threetimes total on Days 0, 3 and 7 with 1 ug of PSMAxCD3 bispecific i.p.Data are expressed as mean (SEM) and were analyzed using analysis ofvariance (ANOVA).

FIGS. 2A-2D show that treatment with a PSMAxCD3 bispecific antibodyinduces transient dose-dependent increase in circulating cytokines,where the cytokine levels (interferon-gamma, IFN-g; tumor necrosisfactor, TNF; interleukin-2, IL-2; and interleukin-6, IL-6) are tested at4 hrs after treatment.

FIGS. 3A-3B illustrate that treatment with PSMAxCD3 bispecific antibodyin a humanized T cell mouse (100 μg/mouse) induces acute increase incytokines (e.g. IFNg) (FIG. 3A) as well as transient decrease incirculating T cells (FIG. 3B).

FIGS. 4A-4C illustrate the effect of PSMAxCD3 bispecific antibodies oneffector T cells in the spleen of the immunocompetent mice.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expression “CD3,” as used herein, refers to an antigen which isexpressed on T cells as part of the multimolecular T cell receptor (TCR)and which consists of a homodimer or heterodimer formed from theassociation of two of four receptor chains: CD3-epsilon, CD3-delta,CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acidsequence as set forth in SEQ ID NO:1649; human CD3-delta comprises theamino acid sequence as set forth in SEQ ID NO:1650. All references toproteins, polypeptides and protein fragments herein are intended torefer to the human version of the respective protein, polypeptide orprotein fragment unless explicitly specified as being from a non-humanspecies. Thus, the expression “CD3” means human CD3 unless specified asbeing from a non-human species, e.g., “mouse CD3,” “monkey CD3,” etc.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodiesand antigen-binding fragments of the present invention may bind solubleCD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

As used herein, the expression “cell surface-expressed CD3” means one ormore CD3 protein(s) that is/are expressed on the surface of a cell invitro or in vivo, such that at least a portion of a CD3 protein isexposed to the extracellular side of the cell membrane and is accessibleto an antigen-binding portion of an antibody. “Cell surface-expressedCD3” includes CD3 proteins contained within the context of a functionalT cell receptor in the membrane of a cell. The expression “cellsurface-expressed CD3” includes CD3 protein expressed as part of ahomodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon,delta/epsilon, and zeta/zeta CD3 dimers). The expression, “cellsurface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon,CD3-delta or CD3-gamma) that is expressed by itself, without other CD3chain types, on the surface of a cell. A “cell surface-expressed CD3”can comprise or consist of a CD3 protein expressed on the surface of acell which normally expresses CD3 protein. Alternatively, “cellsurface-expressed CD3” can comprise or consist of CD3 protein expressedon the surface of a cell that normally does not express human CD3 on itssurface but has been artificially engineered to express CD3 on itssurface.

The expression “PSMA,” as used herein, refers to prostate-specificmembrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is anintegral, non-shed membrane glycoprotein that is highly expressed inprostate epithelial cells and is a cell-surface marker for prostatecancer. The amino acid sequence of human PSMA is set forth in SEQ IDNO:1651.

As used herein, “an antibody that binds PSMA” or an “anti-PSMA antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize PSMA.

The term “antigen-binding molecule” includes antibodies andantigen-binding fragments of antibodies, including, e.g., bispecificantibodies.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., PSMA or CD3). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The VH and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and V_(L) is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-PSMA antibody oranti-CD3 antibody (or antigen-binding portion thereof) may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a VH domain associated with a V_(L)domain, the V_(H) and V_(L) domains may be situated relative to oneanother in any suitable arrangement. For example, the variable regionmay be dimeric and contain V_(H)—V_(H), V_(H)—V_(L) or V_(L)—V_(L)dimers. Alternatively, the antigen-binding fragment of an antibody maycontain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-PSMA monospecificantibodies or anti-PSMA/anti-CD3 bispecific antibodies of the inventionare human antibodies. The term “human antibody”, as used herein, isintended to include antibodies having variable and constant regionsderived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention also includes one-arm antibodies that bind PSMA.As used herein, a “one-arm antibody” means an antigen-binding moleculecomprising a single antibody heavy chain and a single antibody lightchain. The one-arm antibodies of the present invention may comprise anyof the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.

The anti-PSMA or anti-PSMA/anti-CD3 antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes antibodies,and antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-PSMA or anti-PSMA/anti-CD3antibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-PSMA oranti-PSMA/anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acidsequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer,etc. conservative amino acid substitutions relative to any of the HCVR,LCVR, and/or CDR amino acid sequences set forth in Table 1 herein or asdescribed in Tables 12, 14, 15, 18, and 20 herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Germline Mutations

The anti-CD3 antibodies disclosed herein comprise one or more amino acidsubstitutions, insertions and/or deletions in the framework and/or CDRregions of the heavy chain variable domains as compared to thecorresponding germline sequences from which the antibodies were derived.

The present invention also includes antibodies, and antigen-bindingfragments thereof, which are derived from any of the amino acidsequences disclosed herein, wherein one or more amino acids within oneor more framework and/or CDR regions are mutated to the correspondingresidue(s) of the germline sequence from which the antibody was derived,or to the corresponding residue(s) of another human germline sequence,or to a conservative amino acid substitution of the correspondinggermline residue(s) (such sequence changes are referred to hereincollectively as “germline mutations”), and having weak or no detectablebinding to a CD3 antigen. Several such exemplary antibodies thatrecognize CD3 are described in Tables 12 and 18 herein.

Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be tested for one or more desired properties such as, improvedbinding specificity, weak or reduced binding affinity, improved orenhanced pharmacokinetic properties, reduced immunogenicity, etc.Antibodies and antigen-binding fragments obtained in this general mannergiven the guidance of the present disclosure are encompassed within thepresent invention.

The present invention also includes anti-CD3 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-CD3 antibodies having HCVR,LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 orfewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Tables 12, 14, 15, 18, and 20 herein. Theantibodies and bispecific antigen-binding molecules of the presentinvention comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived, while maintaining or improving the desired weak-to-nodetectable binding to CD3 antigen. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein, i.e. the amino acid substitutionmaintains or improves the desired weak to no detectable binding affinityin the case of anti-CD3 binding molecules. Examples of groups of aminoacids that have side chains with similar chemical properties include (1)aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;(2) aliphatic-hydroxyl side chains: serine and threonine; (3)amide-containing side chains: asparagine and glutamine; (4) aromaticside chains: phenylalanine, tyrosine, and tryptophan; (5) basic sidechains: lysine, arginine, and histidine; (6) acidic side chains:aspartate and glutamate, and (7) sulfur-containing side chains arecysteine and methionine. Preferred conservative amino acids substitutiongroups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, andasparagine-glutamine. Alternatively, a conservative replacement is anychange having a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR and/or CDR amino acid sequencethat is substantially identical to any of the HCVR and/or CDR amino acidsequences disclosed herein, while maintaining or improving the desiredweak affinity to CD3 antigen. The term “substantial identity” or“substantially identical,” when referring to an amino acid sequencemeans that two amino acid sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least95% sequence identity, even more preferably at least 98% or 99% sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. In cases where two or moreamino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402.

Once obtained, antigen-binding domains that contain one or more germlinemutations were tested for decreased binding affinity utilizing one ormore in vitro assays. Although antibodies that recognize a particularantigen are typically screened for their purpose by testing for high(i.e. strong) binding affinity to the antigen, the antibodies of thepresent invention exhibit weak binding or no detectable binding.Bispecific antigen-binding molecules comprising one or moreantigen-binding domains obtained in this general manner are alsoencompassed within the present invention and were found to beadvantageous as avidity-driven tumor therapies.

Unexpected benefits, for example, improved pharmacokinetic propertiesand low toxicity to the patient may be realized from the methodsdescribed herein.

Binding Properties of the Antibodies

As used herein, the term “binding” in the context of the binding of anantibody, immunoglobulin, antibody-binding fragment, or Fc-containingprotein to either, e.g., a predetermined antigen, such as a cell surfaceprotein or fragment thereof, typically refers to an interaction orassociation between a minimum of two entities or molecular structures,such as an antibody-antigen interaction.

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore™ 3000 instrument using the antigen as theligand and the antibody, Ig, antibody-binding fragment, or Fc-containingprotein as the analyte (or antiligand). Cell-based binding strategies,such as fluorescent-activated cell sorting (FACS) binding assays, arealso routinely used, and FACS data correlates well with other methodssuch as radioligand competition binding and SPR (Benedict, Calif., JImmunol Methods. 1997, 201(2):223-31; Geuijen, C A, et al. J ImmunolMethods. 2005, 302(1-2):68-77).

Accordingly, the antibody or antigen-binding protein of the inventionbinds to the predetermined antigen or cell surface molecule (receptor)having an affinity corresponding to a K_(D) value that is at leastten-fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein). According to the present invention, the affinity ofan antibody corresponding to a K_(D) value that is equal to or less thanten-fold lower than a non-specific antigen may be considerednon-detectable binding, however such an antibody may be paired with asecond antigen binding arm for the production of a bispecific antibodyof the invention.

The term “K_(D)” (M) refers to the dissociation equilibrium constant ofa particular antibody-antigen interaction, or the dissociationequilibrium constant of an antibody or antibody-binding fragment bindingto an antigen. There is an inverse relationship between K_(D) andbinding affinity, therefore the smaller the K_(D) value, the higher,i.e. stronger, the affinity. Thus, the terms “higher affinity” or“stronger affinity” relate to a higher ability to form an interactionand therefore a smaller K_(D) value, and conversely the terms “loweraffinity” or “weaker affinity” relate to a lower ability to form aninteraction and therefore a larger K_(D) value. In some circumstances, ahigher binding affinity (or K_(D)) of a particular molecule (e.g.antibody) to its interactive partner molecule (e.g. antigen X) comparedto the binding affinity of the molecule (e.g. antibody) to anotherinteractive partner molecule (e.g. antigen Y) may be expressed as abinding ratio determined by dividing the larger K_(D) value (lower, orweaker, affinity) by the smaller K_(D) (higher, or stronger, affinity),for example expressed as 5-fold or 10-fold greater binding affinity, asthe case may be.

The term “k_(d)” (sec-1 or 1/s) refers to the dissociation rate constantof a particular antibody-antigen interaction, or the dissociation rateconstant of an antibody or antibody-binding fragment. Said value is alsoreferred to as the k_(off) value.

The term “k_(a)” (M-1×sec-1 or 1/M) refers to the association rateconstant of a particular antibody-antigen interaction, or theassociation rate constant of an antibody or antibody-binding fragment.

The term “K_(A)” (M-1 or 1/M) refers to the association equilibriumconstant of a particular antibody-antigen interaction, or theassociation equilibrium constant of an antibody or antibody-bindingfragment. The association equilibrium constant is obtained by dividingthe k_(a) by the k_(d).

The term “EC50” or “EC₅₀” refers to the half maximal effectiveconcentration, which includes the concentration of an antibody whichinduces a response halfway between the baseline and maximum after aspecified exposure time. The EC50 essentially represents theconcentration of an antibody where 50% of its maximal effect isobserved. In certain embodiments, the EC₅₀ value equals theconcentration of an antibody of the invention that gives half-maximalbinding to cells expressing CD3 or tumor-associated antigen, asdetermined by e.g. a FACS binding assay. Thus, reduced or weaker bindingis observed with an increased EC₅₀, or half maximal effectiveconcentration value.

In one embodiment, decreased binding can be defined as an increased EC₅₀antibody concentration which enables binding to the half-maximal amountof target cells.

In another embodiment, the EC₅₀ value represents the concentration of anantibody of the invention that elicits half-maximal depletion of targetcells by T cell cytotoxic activity. Thus, increased cytotoxic activity(e.g. T cell-mediated tumor cell killing) is observed with a decreasedEC₅₀, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-PSMA monospecific antibodies oranti-PSMA/anti-CD3 bispecific antibodies of the present invention can belinked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second or additionalbinding specificity.

Use of the expression “anti-CD3 antibody” or “anti-PSMA antibody” hereinis intended to include both monospecific anti-CD3 or anti-PSMAantibodies as well as bispecific antibodies comprising a CD3-binding armand a PSMA-binding arm. Thus, the present invention includes bispecificantibodies wherein one arm of an immunoglobulin binds human CD3, and theother arm of the immunoglobulin is specific for human PSMA. TheCD3-binding arm can comprise any of the HCVR/LCVR or CDR amino acidsequences as set forth in Tables 12, 14, 15, 18, and 20 herein.

In certain embodiments, the CD3-binding arm binds to human CD3 andinduces human T cell activation. In certain embodiments, the CD3-bindingarm binds weakly to human CD3 and induces human T cell activation. Inother embodiments, the CD3-binding arm binds weakly to human CD3 andinduces tumor-associated antigen-expressing cell killing in the contextof a bispecific or multispecific antibody. In other embodiments, theCD3-binding arm binds or associated weakly with human and cynomolgus(monkey) CD3, yet the binding interaction is not detectable by in vitroassays known in the art. The PSMA-binding arm can comprise any of theHCVR/LCVR or CDR amino acid sequences as set forth in Table 1 herein.

According to certain exemplary embodiments, the present inventionincludes bispecific antigen-binding molecules that specifically bind CD3and PSMA. Such molecules may be referred to herein as, e.g.,“anti-CD3/anti-PSMA,” or “anti-CD3×PSMA” or “CD3×PSMA” bispecificmolecules, or other similar terminology (e.g., anti-PSMA/anti-CD3).

The term “PSMA,” as used herein, refers to the human PSMA protein unlessspecified as being from a non-human species (e.g., “mouse PSMA,” “monkeyPSMA,” etc.). The human PSMA protein has the amino acid sequence shownin SEQ ID NO:1651.

The aforementioned bispecific antigen-binding molecules thatspecifically bind CD3 and PSMA may comprise an anti-CD3 antigen-bindingmolecule which binds to CD3 with a weak binding affinity such asexhibiting a K_(D) of greater than about 40 nM, as measured by an invitro affinity binding assay.

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., CD3),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., PSMA).

In certain exemplary embodiments of the present invention, thebispecific antigen-binding molecule is a bispecific antibody. Eachantigen-binding domain of a bispecific antibody comprises a heavy chainvariable domain (HCVR) and a light chain variable domain (LCVR). In thecontext of a bispecific antigen-binding molecule comprising a first anda second antigen-binding domain (e.g., a bispecific antibody), the CDRsof the first antigen-binding domain may be designated with the prefix“A1” and the CDRs of the second antigen-binding domain may be designatedwith the prefix “A2”. Thus, the CDRs of the first antigen-binding domainmay be referred to herein as Al-HCDR1, A1-HCDR2, and A1-HCDR3; and theCDRs of the second antigen-binding domain may be referred to herein asA2-HCDR1, A2-HCDR2, and A2-HCDR3.

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abispecific antigen-binding molecule of the present invention.Alternatively, the first antigen-binding domain and the secondantigen-binding domain may each be connected to a separate multimerizingdomain. The association of one multimerizing domain with anothermultimerizing domain facilitates the association between the twoantigen-binding domains, thereby forming a bispecific antigen-bindingmolecule. As used herein, a “multimerizing domain” is any macromolecule,protein, polypeptide, peptide, or amino acid that has the ability toassociate with a second multimerizing domain of the same or similarstructure or constitution. For example, a multimerizing domain may be apolypeptide comprising an immunoglobulin C_(H)3 domain. A non-limitingexample of a multimerizing component is an Fc portion of animmunoglobulin (comprising a C_(H)2-C_(H)3 domain), e.g., an Fc domainof an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as wellas any allotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of from 1 to about 200 amino acids in lengthcontaining at least one cysteine residue. In other embodiments, themultimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bispecificantigen-binding molecule. Specific exemplary bispecific formats that canbe used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody,IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab² bispecific formats (see,e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,for a review of the foregoing formats).

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q orK) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g.,434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes bispecific antigen-binding moleculescomprising a first C_(H)3 domain and a second Ig C_(H)3 domain, whereinthe first and second Ig C_(H)3 domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No.8,586,713. Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies.

In certain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype. Forexample, a chimeric Fc domain can comprise part or all of a C_(H)2sequence derived from a human IgG1, human IgG2 or human IgG4 C_(H)2region, and part or all of a C_(H)3 sequence derived from a human IgG1,human IgG2 or human IgG4. A chimeric Fc domain can also contain achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” sequence, derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence, derivedfrom a human IgG1, a human IgG2 or a human IgG4 hinge region. Aparticular example of a chimeric Fc domain that can be included in anyof the antigen-binding molecules set forth herein comprises, from N- toC-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4C_(H)2]-[IgG4 C_(H)3]. Another example of a chimeric Fc domain that canbe included in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upper hinge]-[IgG2lower hinge]-[IgG4 C_(H)2]-[IgG1 C_(H)3]. These and other examples ofchimeric Fc domains that can be included in any of the antigen-bindingmolecules of the present invention are described in US Publication2014/0243504, published Aug. 28, 2014, which is herein incorporated inits entirety. Chimeric Fc domains having these general structuralarrangements, and variants thereof, can have altered Fc receptorbinding, which in turn affects Fc effector function.

In certain embodiments, the invention provides an antibody heavy chainwherein the heavy chain constant region (CH) region comprises an aminoacid sequence at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical to any one of SEQ ID NO: 1663, SEQ ID NO: 1664, SEQID NO: 1665, SEQ ID NO: 1666, SEQ ID NO: 1667, SEQ ID NO: 1668, SEQ IDNO: 1669, SEQ ID NO: 1670 SEQ ID NO: 1671 or SEQ ID NO: 1672. In someembodiments, the heavy chain constant region (CH) region comprises anamino acid sequence selected from the group consisting of SEQ ID NO:1663, SEQ ID NO: 1664, SEQ ID NO: 1665, SEQ ID NO: 1666, SEQ ID NO:1667, SEQ ID NO: 1668, SEQ ID NO: 1669, SEQ ID NO: 1670, SEQ ID NO: 1671and SEQ ID NO: 1672.

In other embodiments, the invention provides an antibody heavy chainwherein the Fc domain comprises an amino acid sequence at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identical to any oneof SEQ ID NO: 1673, SEQ ID NO: 1674, SEQ ID NO: 1675, SEQ ID NO: 1676,SEQ ID NO: 1677, SEQ ID NO: 1678, SEQ ID NO: 1679, SEQ ID NO: 1680, SEQID NO: 1681 or SEQ ID NO: 1682. In some embodiments, the Fc domaincomprises an amino acid sequence selected form the group consisting ofSEQ ID NO: 1673, SEQ ID NO: 1674, SEQ ID NO: 1675, SEQ ID NO: 1676, SEQID NO: 1677, SEQ ID NO: 1678, SEQ ID NO: 1679, SEQ ID NO: 1680, SEQ IDNO: 1681 and SEQ ID NO: 1682.

Sequence Variants

The antibodies and bispecific antigen-binding molecules of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. Theantigen-binding molecules of the present invention may compriseantigen-binding domains which are derived from any of the exemplaryamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antigen-binding domain wasoriginally derived. In other embodiments, only certain residues aremutated back to the original germline sequence, e.g., only the mutatedresidues found within the first 8 amino acids of FR1 or within the last8 amino acids of FR4, or only the mutated residues found within CDR1,CDR2 or CDR3. In other embodiments, one or more of the framework and/orCDR residue(s) are mutated to the corresponding residue(s) of adifferent germline sequence (i.e., a germline sequence that is differentfrom the germline sequence from which the antigen-binding domain wasoriginally derived). Furthermore, the antigen-binding domains maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germlinesequence while certain other residues that differ from the originalgermline sequence are maintained or are mutated to the correspondingresidue of a different germline sequence. Once obtained, antigen-bindingdomains that contain one or more germline mutations can be easily testedfor one or more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Bispecific antigen-binding molecules comprising oneor more antigen-binding domains obtained in this general manner areencompassed within the present invention.

The present invention also includes antigen-binding molecules whereinone or both antigen-binding domains comprise variants of any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein having oneor more conservative substitutions. For example, the present inventionincludes antigen-binding molecules comprising an antigen-binding domainhaving HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 orfewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. A “conservative amino acid substitution” isone in which an amino acid residue is substituted by another amino acidresidue having a side chain (R group) with similar chemical properties(e.g., charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. Examples of groups of amino acids that have side chains withsimilar chemical properties include (1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl sidechains: serine and threonine; (3) amide-containing side chains:asparagine and glutamine; (4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, andhistidine; (6) acidic side chains: aspartate and glutamate, and (7)sulfur-containing side chains are cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443-1445, herein incorporated by reference.A “moderately conservative” replacement is any change having anonnegative value in the PAM250 log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR, LCVR, and/or CDR amino acidsequence that is substantially identical to any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein. The term “substantialidentity” or “substantially identical,” when referring to an amino acidsequence means that two amino acid sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 95% sequence identity, even more preferably at least 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. In cases where two ormore amino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, herein incorporated by reference.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-PSMA antibodies, andanti-CD3/anti-PSMA bispecific antigen-binding molecules, withpH-dependent binding characteristics. For example, an anti-PSMA antibodyof the present invention may exhibit reduced binding to PSMA at acidicpH as compared to neutral pH. Alternatively, anti-PSMA antibodies of theinvention may exhibit enhanced binding to PSMA at acidic pH as comparedto neutral pH. The expression “acidic pH” includes pH values less thanabout 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6,5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, orless. As used herein, the expression “neutral pH” means a pH of about7.0 to about 7.4. The expression “neutral pH” includes pH values ofabout 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding . . . at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of theantibody binding to its antigen at acidic pH to the K_(D) value of theantibody binding to its antigen at neutral pH (or vice versa). Forexample, an antibody or antigen-binding fragment thereof may be regardedas exhibiting “reduced binding to PSMA at acidic pH as compared toneutral pH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-PSMAantibodies, and anti-CD3/anti-PSMA bispecific antigen-binding molecules,are provided comprising an Fc domain comprising one or more mutationswhich enhance or diminish antibody binding to the FcRn receptor, e.g.,at acidic pH as compared to neutral pH. For example, the presentinvention includes antibodies comprising a mutation in the C_(H)2 or aC_(H)3 region of the Fc domain, wherein the mutation(s) increases theaffinity of the Fc domain to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0). Such mutationsmay result in an increase in serum half-life of the antibody whenadministered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the present invention includes anti-PSMA antibodies, andanti-CD3/anti-PSMA bispecific antigen-binding molecules, comprising anFc domain comprising one or more pairs or groups of mutations selectedfrom the group consisting of: 250Q and 248L (e.g., T250Q and M248L);252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g.,M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Allpossible combinations of the foregoing Fc domain mutations, and othermutations within the antibody variable domains disclosed herein, arecontemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and BispecificAntigen-Binding Molecules

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human PSMA with high affinity (e.g., sub-nanomolarK_(D) values).

According to certain embodiments, the present invention includesantibodies and antigen-binding fragments of antibodies that bind humanPSMA (e.g., at 37° C.) with a K_(D) of less than about 80 nM as measuredby surface plasmon resonance, e.g., using an assay format as defined inExample 3 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind PSMA with aK_(D) of less than about 5 nM, less than about 2 nM, less than about 1nM, less than about 800 pM, less than about 600 pM, less than about 500pM, less than about 400 pM, less than about 300 pM, less than about 200pM, less than about 180 pM, less than about 160 pM, less than about 140pM, less than about 120 pM, less than about 100 pM, less than about 80pM, less than about 60 pM, less than about 40 pM, less than about 20 pM,or less than about 10 pM, as measured by surface plasmon resonance,e.g., using an assay format as defined in Example 3 herein (e.g.,mAb-capture or antigen-capture format), or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind PSMA with a dissociative half-life (t½) ofgreater than about 1 minute or greater than about 10 minutes as measuredby surface plasmon resonance at 37° C., e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay. Incertain embodiments, the antibodies or antigen-binding fragments of thepresent invention bind PSMA with a t½ of greater than about 20 minutes,greater than about 30 minutes, greater than about 40 minutes, greaterthan about 50 minutes, greater than about 60 minutes, greater than about70 minutes, greater than about 80 minutes, greater than about 90minutes, greater than about 100 minutes, greater than about 200 minutes,greater than about 300 minutes, greater than about 400 minutes, greaterthan about 500 minutes, greater than about 600 minutes, greater thanabout 700 minutes, greater than about 800 minutes, greater than about900 minutes, greater than about 1000 minutes, or greater than about 1100minutes, as measured by surface plasmon resonance at 25° C. or 37° C.,e.g., using an assay format as defined in Example 3 herein (e.g.,mAb-capture or antigen-capture format), or a substantially similarassay. The present invention includes bispecific antigen-bindingmolecules (e.g., bispecific antibodies) which are capable ofsimultaneously binding to human CD3 and human PSMA. According to certainembodiments, the bispecific antigen-binding molecules of the inventionspecifically interact with cells that express CD3 and/or PSMA. Theextent to which a bispecific antigen-binding molecule binds cells thatexpress CD3 and/or PSMA can be assessed by fluorescence activated cellsorting (FACS), as illustrated in Example 5 herein. For example, thepresent invention includes bispecific antigen-binding molecules whichspecifically bind human T-cell lines which express CD3 (such cell linesdo not express PSMA, e.g., Jurkat) and/or human lines which express PSMA(such cell lines do not express CD3, e.g., B16F10.9/hPSMA or 22RV1). Thepresent invention includes bispecific antigen-binding molecules whichbind any of the aforementioned cells and cell lines with an EC₅₀ valueof about 80 nM, or less, as determined using a FACS assay as set forthin Example 5 or a substantially similar assay.

The present invention also includes anti-CD3/anti-PSMA bispecificantigen-binding molecules which bind to CD3-expressing human T-cells(e.g., Jurkat) and/or PSMA-expressing cells with an EC₅₀ value ofbetween 1.0 pM and 1000 nM. In certain embodiments, theanti-CD3/anti-PSMA bispecific antigen-binding molecules bind toCD3-expressing human T-cells with an EC50 value of between 1 nM and 60nM. For example, the present invention includes anti-CD3/anti-PSMAbispecific antigen-binding molecules which bind to CD3-expressing humanT-cells (e.g., Jurkat) and/or PSMA-expressing cells with an EC₅₀ valueof about 1 pM. about 10 pM, about 100 pM, about 500 pM, about 1 nM,about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40nM, about 50 nM about 60 nM, about 70 nM, about 80 nM, about 90 nM,about 100 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM,about 1000 nM, or more.

The present invention also includes anti-CD3/anti-PSMA bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of: (a) inhibiting tumor growth inimmunocompromised mice bearing human prostate cancer xenografts; (b)inhibiting tumor growth in immunocompetent mice bearing human prostatecancer xenografts; (c) suppressing tumor growth of established tumors inimmunocompromised mice bearing human prostate cancer xenografts; and (d)reducing tumor growth of established tumors in immunocompetent micebearing human prostate cancer xenografts (see, e.g., Example 8).

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with high affinity. The present inventionalso includes antibodies and antigen-binding fragments thereof that bindhuman CD3 with medium or low affinity, depending on the therapeuticcontext and particular targeting properties that are desired. Forexample, in the context of a bispecific antigen-binding molecule,wherein one arm binds CD3 and another arm binds a target antigen (e.g.,PSMA), it may be desirable for the target antigen-binding arm to bindthe target antigen with high affinity while the anti-CD3 arm binds CD3with only moderate or low affinity. In this manner, preferentialtargeting of the antigen-binding molecule to cells expressing the targetantigen may be achieved while avoiding general/untargeted CD3 bindingand the consequent adverse side effects associated therewith.

The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies) which are capable of simultaneouslybinding to human CD3 and a human PSMA. The binding arm that interactswith cells that express CD3 may have weak to no detectable binding asmeasured in a suitable in vitro binding assay. The extent to which abispecific antigen-binding molecule binds cells that express CD3 and/orPSMA can be assessed by fluorescence activated cell sorting (FACS), asillustrated in Example 5 herein.

For example, the present invention includes antibodies, antigen-bindingfragments, and bispecific antibodies thereof which specifically bindhuman T-cell lines which express CD3 but do not express PSMA (e.g.,Jurkat), primate T-cells (e.g., cynomolgus peripheral blood mononuclearcells [PBMCs]), and/or PSMA-expressing cells. The present inventionincludes bispecific antigen-binding molecules which bind any of theaforementioned T cells and T cell lines with an EC₅₀ value of from about1.8×10⁻⁸ (18 nM) to about 2.1×10⁻⁷ (210 nM), or more (i.e. weakeraffinity), or EC₅₀ is undetectable, as determined using a FACS bindingassay as set forth in Example 5 or a substantially similar assay. Incertain embodiments, the antibodies, antigen-binding fragments, andbispecific antibodies of the present invention bind CD3 with an EC50 ofgreater than about 30 nM, greater than about 40 nM, greater than about45 nM, greater than about 50 nM, greater than about 55 nM, greater thanabout 60 nM, greater than about 65 nM, greater than about 70 nM, greaterthan about 75 nM, at least 80 nM, greater than about 90 nM, greater thanabout 100 nM, greater than about 110 nM, at least 120 nM, greater thanabout 130 nM, greater than about 140 nM, greater than about 150 nM, atleast 160 nM, greater than about 170 nM, greater than about 180 nM,greater than about 190 nM, greater than about 200 nM, greater than about250 nM, greater than about 300 nM, greater than about 1 μM, greater thanabout 2 μM, or greater than about 3 μM, or no detectable affinity, asmeasured by FACS binding, e.g., using an assay format as defined inExample 5 herein, or a substantially similar assay.

The present invention also includes antibodies, antigen-bindingfragments, and bispecific antibodies thereof which bind toPSMA-expressing cells and cell lines, with an EC₅₀ value of less than orequal to 5.6 nM (5.6×10⁻⁹), as determined using a FACS binding assay asset forth in Example 5 or a substantially similar assay.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 with weak (i.e.low) or even no detectable affinity. According to certain embodiments,the present invention includes antibodies and antigen-binding fragmentsof antibodies that bind human CD3 (e.g., at 37° C.) with a K_(D) ofgreater than about 11 nM as measured by surface plasmon resonance, e.g.,using an assay format as defined in Example 6 herein. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention bind CD3 with a K_(D) of greater than about 15 nM, greaterthan about 20 nM, greater than about 25 nM, greater than about 30 nM,greater than about 35 nM, greater than about 40 nM, greater than about45 nM, greater than about 50 nM, greater than about 55 nM, greater thanabout 60 nM, greater than about 65 nM, greater than about 70 nM, greaterthan about 75 nM, at least 80 nM, greater than about 90 nM, greater thanabout 100 nM, greater than about 110 nM, at least 120 nM, greater thanabout 130 nM, greater than about 140 nM, greater than about 150 nM, atleast 160 nM, greater than about 170 nM, greater than about 180 nM,greater than about 190 nM, greater than about 200 nM, greater than about250 nM, greater than about 300 nM, greater than about 1 pM, greater thanabout 2 μM, or greater than about 3 μM, or no detectable affinity, asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 6 herein (e.g., mAb-capture or antigen-captureformat), or a substantially similar assay.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind monkey (i.e. cynomolgus) CD3with weak (i.e. low) or even no detectable affinity. According tocertain embodiments, the present invention includes antibodies,antigen-binding fragments, and bispecific antibodies thereof that bindhuman CD3 (e.g., at 37° C.) with a K_(D) of greater than about 10 nM asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 6 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind CD3 with a K_(D)of greater than about 15 nM, greater than about 20 nM, greater thanabout 25 nM, greater than about 30 nM, greater than about 35 nM, greaterthan about 40 nM, greater than about 45 nM, greater than about 50 nM,greater than about 55 nM, greater than about 60 nM, greater than about65 nM, greater than about 70 nM, greater than about 75 nM, at least 80nM, greater than about 90 nM, greater than about 100 nM, greater thanabout 110 nM, at least 120 nM, greater than about 130 nM, greater thanabout 140 nM, greater than about 150 nM, at least 160 nM, greater thanabout 170 nM, greater than about 180 nM, greater than about 190 nM,greater than about 200 nM, greater than about 250 nM, greater than about300 nM, greater than about 1 μM, greater than about 2 μM, or greaterthan about 3 μM, or no detectable affinity, as measured by surfaceplasmon resonance, e.g., using an assay format as defined in Example 6herein (e.g., mAb-capture or antigen-capture format), or a substantiallysimilar assay.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 and induce T cellactivation. For example, the present invention includes anti-CD3antibodies that induce human T cell activation with an EC₅₀ value ofless than about 113 pM, as measured by an in vitro T cell activationassay, e.g., using the assay format as defined in Example 7 herein[e.g., assessing the percent activated (CD69+) cells out of total Tcells (CD2+) in the presence of anti-CD3 antibodies], or a substantiallysimilar assay that assesses T cell in their activated state. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention induce human T cell activation [e.g., percent activated(CD69+) T cells] with an EC₅₀ value of less than about 100 pM, less thanabout 50 pM, less than about 20 pM, less than about 19 pM, less thanabout 18 pM, less than about 17 pM, less than about 16 pM, less thanabout 15 pM, less than about 14 pM, less than about 13 pM, less thanabout 12 pM, less than about 11 pM, less than about 10 pM, less thanabout 9 pM, less than about 8 pM, less than about 7 pM, less than about6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM,less than about 2 pM, or less than about 1 pM, as measured by an invitro T cell activation assay, e.g., using the assay format as definedin Example 7 herein, or a substantially similar assay. Anti-CD3antibodies that have weak or no detectable binding to CD3 have theability to induce T cell activation with high potency (i.e. pM range),despite having weak or no detectable binding affinity to CD3, asexemplified in Example 7 herein.

The present invention also includes antibodies, antigen-bindingfragments, and bispecific antibodies that bind human CD3 and induce Tcell-mediated killing of tumor antigen-expressing cells. For example,the present invention includes anti-CD3 antibodies that induce Tcell-mediated killing of tumor cells with an EC₅₀ of less than about 1.3nM, as measured in an in vitro T cell-mediated tumor cell killing assay,e.g., using the assay format as defined in Example 7 herein (e.g.,assessing the extent of PSMA-expressing cell killing by human PBMCs inthe presence of anti-CD3 antibodies), or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention induce T cell-mediated tumor cell killing (e.g.,PBMC-mediated killing of C4-2, 22Rv1 and TRAMPC2_PSMA cells) with anEC₅₀ value of less than about 1 nM, less than about 400 pM, less thanabout 250 pM, less than about 100 pM, less than about 50 pM, less thanabout 40 pM, less than about 30 pM, less than about 20 pM, less thanabout 10 pM, less than about 9 pM, less than about 8 pM, less than about7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM,less than about 3 pM, less than about 2 pM, or less than about 1 pM, asmeasured by an in vitro T cell-mediated tumor cell killing assay, e.g.,using the assay format as defined in Example 7 herein, or asubstantially similar assay. The present invention also includesantibodies, antigen-binding fragments, and bispecific antibodies thatbind human and/or monkey (i.e. cynomolgus) CD3 with weak (i.e. low) oreven no detectable affinity (i.e. do not bind or exhibit no detectableaffinity) and induce T cell-mediated killing of tumor antigen-expressingcells.

The present invention also includes antibodies, antigen-bindingfragments, and bispecific antibodies that bind CD3 with a dissociativehalf-life (t½) of less than about 10 minutes as measured by surfaceplasmon resonance at 25° C. or 37° C., e.g., using an assay format asdefined in Example 6 herein, or a substantially similar assay. Incertain embodiments, the antibodies or antigen-binding fragments of thepresent invention bind CD3 with a t½ of less than about 9 minutes, ofless than about 8 minutes, of less than about 7 minutes, of less thanabout 6 minutes, of less than about 5 minutes, of less than about 4minutes, of less than about 3 minutes, of less than about 2 minutes, ofless than about 1.9 minutes, or less than about 1.8 minutes, or exhibitvery weak or no detectable binding as measured by surface plasmonresonance at 25° C. or 37° C., e.g., using an assay format as defined inExample 6 herein (e.g., mAb-capture or antigen-capture format), or asubstantially similar assay.

The anti-CD3/anti-PSMA bispecific antigen-binding molecules of thepresent invention may additionally exhibit one or more characteristicsselected from the group consisting of: (a) inducing PBMC proliferationin vitro; (b) activating T-cells via inducing IFN-gamma release and CD25up-regulation in human whole blood; and (c) inducing T-cell mediatedcytotoxicity on anti-PSMA-resistant cell lines.

The present invention includes anti-CD3/anti-PSMA bispecificantigen-binding molecules which are capable of depleting tumorantigen-expressing cells in a subject (see, e.g., Example 8). Forexample, according to certain embodiments, anti-CD3/anti-PSMA bispecificantigen-binding molecules are provided, wherein a single administrationof 1 μg, or 10 μg, or 100 μg of the bispecific antigen-binding moleculeto a subject (e.g., at a dose of about 0.1 mg/kg, about 0.08 mg/kg,about 0.06 mg/kg about 0.04 mg/kg, about 0.04 mg/kg, about 0.02 mg/kg,about 0.01 mg/kg, or less) causes a reduction in the number ofPSMA-expressing cells in the subject (e.g., tumor growth in the subjectis suppressed or inhibited) below detectable levels. In certainembodiments, a single administration of the anti-CD3/anti-PSMAbispecific antigen-binding molecule at a dose of about 0.4 mg/kg causesa reduction in tumor growth in the subject below detectable levels byabout day 7, about day 6, about day 5, about day 4, about day 3, aboutday 2, or about day 1 after administration of the bispecificantigen-binding molecule to the subject. According to certainembodiments, a single administration of an anti-CD3/anti-PSMA bispecificantigen-binding molecule of the invention, at a dose of at least about0.01 mg/kg, causes the number of PSMA-expressing tumor cells to remainbelow detectable levels until at least about 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days ormore, following the administration. As used herein, the expression“below detectable levels” means that no tumor cells can be directly orindirectly detected growing subcutaneously in a subject using standardcaliper measurement methods, e.g., as set forth in Example 8, herein.

The present invention also includes anti-CD3/anti-PSMA bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of: (a) inhibiting tumor growth inimmunocompromised mice bearing human prostate cancer xenografts; (b)inhibiting tumor growth in immunocompetent mice bearing human prostatecancer xenografts; (c) suppressing tumor growth of established tumors inimmunocompromised mice bearing human prostate cancer xenografts; and (d)reducing tumor growth of established tumors in immunocompetent micebearing human prostate cancer xenografts (see, e.g., Example 8). Thepresent invention also includes anti-CD3/anti-PSMA bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of: (a) induce transientdose-dependent increases in circulating cytokines, (b) induce transientincreases in circulating T cells, and (c) do not deplete effector T cellcells (e.g. CD4+ T cells, CD8+ T cells, and regulatory T cells, i.e.Tregs).

Epitope Mapping and Related Technologies

The epitope on CD3 and/or PSMA to which the antigen-binding molecules ofthe present invention bind may consist of a single contiguous sequenceof 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) amino acids of a CD3 or PSMA protein. Alternatively,the epitope may consist of a plurality of non-contiguous amino acids (oramino acid sequences) of CD3 or PSMA. The antibodies of the inventionmay interact with amino acids contained within a single CD3 chain (e.g.,CD3-epsilon, CD3-delta or CD3-gamma), or may interact with amino acidson two or more different CD3 chains. The term “epitope,” as used herein,refers to an antigenic determinant that interacts with a specificantigen binding site in the variable region of an antibody moleculeknown as a paratope. A single antigen may have more than one epitope.Thus, different antibodies may bind to different areas on an antigen andmay have different biological effects. Epitopes may be eitherconformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstances, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of an antibody“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques include, e.g., routine cross-blockingassay such as that described Antibodies, Harlow and Lane (Cold SpringHarbor Press, Cold Spring Harb., NY), alanine scanning mutationalanalysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol248:443-463), and peptide cleavage analysis. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer, 2000, Protein Science 9:487-496).Another method that can be used to identify the amino acids within apolypeptide with which an antigen-binding domain of an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

The present invention further includes anti-PSMA antibodies that bind tothe same epitope as any of the specific exemplary antibodies describedherein (e.g. antibodies comprising any of the amino acid sequences asset forth in Table 1 herein). Likewise, the present invention alsoincludes anti-PSMA antibodies that compete for binding to PSMA with anyof the specific exemplary antibodies described herein (e.g. antibodiescomprising any of the amino acid sequences as set forth in Table 1herein).

The present invention also includes bispecific antigen-binding moleculescomprising a first antigen-binding domain that specifically binds humanCD3 and/or cynomolgus CD3 with low or detectable binding affinity, and asecond antigen binding domain that specifically binds human PSMA,wherein the first antigen-binding domain binds to the same epitope onCD3 as any of the specific exemplary CD3-specific antigen-bindingdomains described herein, and/or wherein the second antigen-bindingdomain binds to the same epitope on PSMA as any of the specificexemplary PSMA-specific antigen-binding domains described herein.

Likewise, the present invention also includes bispecific antigen-bindingmolecules comprising a first antigen-binding domain that specificallybinds human CD3, and a second antigen binding domain that specificallybinds human PSMA, wherein the first antigen-binding domain competes forbinding to CD3 with any of the specific exemplary CD3-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain competes for binding to PSMA with any of thespecific exemplary PSMA-specific antigen-binding domains describedherein.

One can easily determine whether a particular antigen-binding molecule(e.g., antibody) or antigen-binding domain thereof binds to the sameepitope as, or competes for binding with, a reference antigen-bindingmolecule of the present invention by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope on PSMA (or CD3) as a reference bispecific antigen-bindingmolecule of the present invention, the reference bispecific molecule isfirst allowed to bind to a PSMA protein (or CD3 protein). Next, theability of a test antibody to bind to the PSMA (or CD3) molecule isassessed. If the test antibody is able to bind to PSMA (or CD3)following saturation binding with the reference bispecificantigen-binding molecule, it can be concluded that the test antibodybinds to a different epitope of PSMA (or CD3) than the referencebispecific antigen-binding molecule. On the other hand, if the testantibody is not able to bind to the PSMA (or CD3) molecule followingsaturation binding with the reference bispecific antigen-bindingmolecule, then the test antibody may bind to the same epitope of PSMA(or CD3) as the epitope bound by the reference bispecificantigen-binding molecule of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference bispecific antigen-binding molecule or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore™,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antigen-binding proteins bind to the same (oroverlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess ofone antigen-binding protein inhibits binding of the other by at least50% but preferably 75%, 90% or even 99% as measured in a competitivebinding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antigen-binding proteins aredeemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantigen-binding protein reduce or eliminate binding of the other. Twoantigen-binding proteins are deemed to have “overlapping epitopes” ifonly a subset of the amino acid mutations that reduce or eliminatebinding of one antigen-binding protein reduce or eliminate binding ofthe other.

To determine if an antibody or antigen-binding domain thereof competesfor binding with a reference antigen-binding molecule, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding molecule is allowedto bind to a PSMA protein (or CD3 protein) under saturating conditionsfollowed by assessment of binding of the test antibody to the PSMA (orCD3) molecule. In a second orientation, the test antibody is allowed tobind to a PSMA (or CD3) molecule under saturating conditions followed byassessment of binding of the reference antigen-binding molecule to thePSMA (or CD3) molecule. If, in both orientations, only the first(saturating) antigen-binding molecule is capable of binding to the PSMA(or CD3) molecule, then it is concluded that the test antibody and thereference antigen-binding molecule compete for binding to PSMA (or CD3).As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antigen-bindingmolecule may not necessarily bind to the same epitope as the referenceantibody, but may sterically block binding of the reference antibody bybinding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,two different antigen-binding domains, specific for two differentantigens (e.g., CD3 and PSMA), can be appropriately arranged relative toone another to produce a bispecific antigen-binding molecule of thepresent invention using routine methods. (A discussion of exemplarybispecific antibody formats that can be used to construct the bispecificantigen-binding molecules of the present invention is provided elsewhereherein). In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains) of the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making such antibodiesare well known in the art. For example, one or more of the heavy and/orlight chains of the bispecific antigen-binding molecules of the presentinvention can be prepared using VELOCIMMUNE™ technology. UsingVELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD3 or PSMA) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

Genetically engineered animals may be used to make human bispecificantigen-binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human bispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454 for a detailed discussion of suchengineered mice and the use thereof to produce bispecificantigen-binding molecules).

Bioequivalents

The present invention encompasses antigen-binding molecules having aminoacid sequences that vary from those of the exemplary molecules disclosedherein but that retain the ability to bind CD3 and/or PSMA. Such variantmolecules may comprise one or more additions, deletions, orsubstitutions of amino acids when compared to parent sequence, butexhibit biological activity that is essentially equivalent to that ofthe described bispecific antigen-binding molecules.

The present invention includes antigen-binding molecules that arebioequivalent to any of the exemplary antigen-binding molecules setforth herein. Two antigen-binding proteins, or antibodies, areconsidered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledoes or multiple dose. Some antigen-binding proteins will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antigen-binding protein.

Bioequivalent variants of the exemplary bispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antigen-binding proteinsmay include variants of the exemplary bispecific antigen-bindingmolecules set forth herein comprising amino acid changes which modifythe glycosylation characteristics of the molecules, e.g., mutationswhich eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, antigen-bindingmolecules are provided which bind to human CD3 but not to CD3 from otherspecies. Also provided are antigen-binding molecules which bind to humanPSMA but not to PSMA from other species. The present invention alsoincludes antigen-binding molecules that bind to human CD3 and to CD3from one or more non-human species; and/or antigen-binding moleculesthat bind to human PSMA and to PSMA from one or more non-human species.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provided which bind to human CD3 and/orhuman PSMA and may bind or not bind, as the case may be, to one or moreof mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CD3and/or PSMA. For example, in a particular exemplary embodiment of thepresent invention bispecific antigen-binding molecules are providedcomprising a first antigen-binding domain that binds human CD3 andcynomolgus CD3, and a second antigen-binding domain that specificallybinds human PSMA.

Immunoconjugates

The present invention encompasses antigen-binding molecules conjugatedto a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe antigen-binding molecules of the present invention. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area. Whena bispecific antigen-binding molecule of the present invention is usedfor therapeutic purposes in an adult patient, it may be advantageous tointravenously administer the bispecific antigen-binding molecule of thepresent invention normally at a single dose of about 0.01 to about 20mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 toabout 5, or about 0.05 to about 3 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering a bispecific antigen-binding molecule may be determinedempirically; for example, patient progress can be monitored by periodicassessment, and the dose adjusted accordingly. Moreover, interspeciesscaling of dosages can be performed using well-known methods in the art(e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-PSMA antibody or antigen-binding fragment thereof, or a bispecificantigen-binding molecule that specifically binds CD3 and PSMA. Thetherapeutic composition can comprise any of the antibodies or bispecificantigen-binding molecules as disclosed herein and a pharmaceuticallyacceptable carrier or diluent. As used herein, the expression “a subjectin need thereof” means a human or non-human animal that exhibits one ormore symptoms or indicia of cancer (e.g., a subject expressing a tumoror suffering from any of the cancers mentioned herein below), or whootherwise would benefit from an inhibition or reduction in PSMA activityor a depletion of PSMA+ cells (e.g., prostate cancer cells).

The antibodies and bispecific antigen-binding molecules of the invention(and therapeutic compositions comprising the same) are useful, interalia, for treating any disease or disorder in which stimulation,activation and/or targeting of an immune response would be beneficial.In particular, the anti-PSMA antibodies or the anti-CD3/anti-PSMAbispecific antigen-binding molecules of the present invention may beused for the treatment, prevention and/or amelioration of any disease ordisorder associated with or mediated by PSMA expression or activity orthe proliferation of PSMA+ cells. The mechanism of action by which thetherapeutic methods of the invention are achieved include killing of thecells expressing PSMA in the presence of effector cells, for example, byCDC, apoptosis, ADCC, phagocytosis, or by a combination of two or moreof these mechanisms. Cells expressing PSMA which can be inhibited orkilled using the bispecific antigen-binding molecules of the inventioninclude, for example, prostate tumor cells.

The antigen-binding molecules of the present invention may be used totreat, e.g., primary and/or metastatic tumors arising in thegastrointestinal tract, prostate, kidney, and/or bladder. In certainembodiments, the bispecific antigen-binding molecules of the inventionare used to treat one or more of the following cancers: clear cell renalcell carcinoma, chromophobe renal cell carcinoma, (renal) oncocytoma,(renal) transitional cell carcinoma, prostate cancer, colorectal cancer,gastric cancer, urothelial carcinoma, (bladder) adenocarcinoma, or(bladder) small cell carcinoma. According to certain embodiments of thepresent invention, the anti-PSMA antibodies or anti-PSMA/anti-CD3bispecific antibodies are useful for treating a patient afflicted with acastrate-resistant prostate cancer. According to other relatedembodiments of the invention, methods are provided comprisingadministering an anti-PSMA antibody or an anti-CD3/anti-PSMA bispecificantigen-binding molecule as disclosed herein to a patient who isafflicted with a castrate-resistant prostate cancer. Analytic/diagnosticmethods known in the art, such as tumor scanning, etc., may be used toascertain whether a patient harbors a tumor that is castrate-resistant.

The present invention also includes methods for treating residual cancerin a subject. As used herein, the term “residual cancer” means theexistence or persistence of one or more cancerous cells in a subjectfollowing treatment with an anti-cancer therapy.

According to certain aspects, the present invention provides methods fortreating a disease or disorder associated with PSMA expression (e.g.,prostate cancer) comprising administering one or more of the anti-PSMAor bispecific antigen-binding molecules described elsewhere herein to asubject after the subject has been determined to have prostate cancer(e.g., castrate-resistant prostate cancer). For example, the presentinvention includes methods for treating prostate cancer comprisingadministering an anti-PSMA antibody or an anti-CD3/anti-PSMA bispecificantigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6months, 8 months, 1 year, or more after the subject has received hormonetherapy (e.g., anti-androgen therapy).

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary antibodiesand bispecific antigen-binding molecules described herein in combinationwith one or more additional therapeutic agents. Exemplary additionaltherapeutic agents that may be combined with or administered incombination with an antigen-binding molecule of the present inventioninclude, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody [e.g.,cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g.,gefitinib or erlotinib]), an antagonist of another EGFR family membersuch as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 oranti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4activity), an antagonist of EGFRvIII (e.g., an antibody thatspecifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMETantibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-rafinhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-αinhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g.,an anti-PDGFR-β antibody), a VEGF antagonist (e.g., a VEGF-Trap, see,e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a“VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), a FOLH1 (PSMA) antagonist, a PRLRantagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist(e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., ananti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), auroplakin antagonist (e.g., an anti-uroplakin antibody), etc. Otheragents that may be beneficially administered in combination with theantigen-binding molecules of the invention include cytokine inhibitors,including small-molecule cytokine inhibitors and antibodies that bind tocytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11,IL-12, IL-13, IL-17, IL-18, or to their respective receptors. Thepharmaceutical compositions of the present invention (e.g.,pharmaceutical compositions comprising an anti-CD3/anti-PSMA bispecificantigen-binding molecule as disclosed herein) may also be administeredas part of a therapeutic regimen comprising one or more therapeuticcombinations selected from “ICE”: ifosfamide (e.g., Ifex®), carboplatin(e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®,VP-16); “DHAP”: dexamethasone (e.g., Decadron®), cytarabine (e.g.,Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g.,Platinol®-AQ); and “ESHAP”: etoposide (e.g., Etopophos®, Toposar®,VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dosecytarabine, cisplatin (e.g., Platinol®-AQ).

The present invention also includes therapeutic combinations comprisingany of the antigen-binding molecules mentioned herein and an inhibitorof one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII,cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1,STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementionedcytokines, wherein the inhibitor is an aptamer, an antisense molecule, aribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment(e.g., Fab fragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv;dAb fragment; or other engineered molecules, such as diabodies,triabodies, tetrabodies, minibodies and minimal recognition units). Theantigen-binding molecules of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids and/or NSAIDs. The antigen-binding moleculesof the invention may also be administered as part of a treatment regimenthat also includes radiation treatment and/or conventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan antigen-binding molecule of the present invention; (for purposes ofthe present disclosure, such administration regimens are considered theadministration of an antigen-binding molecule “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which anantigen-binding molecule of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an antigen-binding molecule (e.g., an anti-PSMA antibody or abispecific antigen-binding molecule that specifically binds PSMA andCD3) may be administered to a subject over a defined time course. Themethods according to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an antigen-binding moleculeof the invention. As used herein, “sequentially administering” meansthat each dose of an antigen-binding molecule is administered to thesubject at a different point in time, e.g., on different days separatedby a predetermined interval (e.g., hours, days, weeks or months). Thepresent invention includes methods which comprise sequentiallyadministering to the patient a single initial dose of an antigen-bindingmolecule, followed by one or more secondary doses of the antigen-bindingmolecule, and optionally followed by one or more tertiary doses of theantigen-binding molecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of an antigen-binding molecule contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule (e.g., an anti-PSMA antibody or abispecific antigen-binding molecule that specifically binds PSMA andCD3). For example, in certain embodiments, only a single secondary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to thepatient. Likewise, in certain embodiments, only a single tertiary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to thepatient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-PSMA antibodies of the present invention may also be used todetect and/or measure PSMA, or PSMA-expressing cells in a sample, e.g.,for diagnostic purposes. For example, an anti-PSMA antibody, or fragmentthereof, may be used to diagnose a condition or disease characterized byaberrant expression (e.g., over-expression, under-expression, lack ofexpression, etc.) of PSMA. Exemplary diagnostic assays for PSMA maycomprise, e.g., contacting a sample, obtained from a patient, with ananti-PSMA antibody of the invention, wherein the anti-PSMA antibody islabeled with a detectable label or reporter molecule. Alternatively, anunlabeled anti-PSMA antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Anotherexemplary diagnostic use of the anti-PSMA antibodies of the inventionincludes ⁸⁹Zr-labeled, such as ⁸⁹Zr-desferrioxamine-labeled, antibodyfor the purpose of noninvasive identification and tracking of tumorcells in a subject (e.g. positron emission tomography (PET) imaging).(See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan. 1; 76(1):73-82; andAzad, B B. et al. Oncotarget. 2016 Mar. 15; 7(11):12344-58.) Specificexemplary assays that can be used to detect or measure PSMA in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in PSMA diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of PSMA protein, orfragments thereof, under normal or pathological conditions. Generally,levels of PSMA in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal PSMA levels or activity) will be measured to initiallyestablish a baseline, or standard, level of PSMA. This baseline level ofPSMA can then be compared against the levels of PSMA measured in samplesobtained from individuals suspected of having a PSMA related disease(e.g., a tumor containing PSMA-expressing cells) or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Generation of Anti-PSMA Antibodies

Anti-PSMA antibodies were obtained by immunizing a genetically modifiedmouse with a human PSMA antigen or by immunizing an engineered mousecomprising DNA encoding human immunoglobulin heavy and kappa light chainvariable regions with a human PSMA antigen.

Mice were immunized with human prostate cancer cells (LNCaP, ATTC®,Manassas, Va., USA) expressing human PSMA (SEQ ID NO:1651;UniProtKB/Swiss-Prot. No. Q04609). Following immunization, splenocyteswere harvested from each mouse and either (1) fused with mouse myelomacells to preserve their viability and form hybridoma cells and screenedfor PSMA specificity, or (2) B-cell sorted (as described in US2007/0280945A1) using a human PSMA with an N-terminal 6-His tag (R&D,Cat #4234-ZN) as the sorting reagent that binds and identifies reactiveantibodies (antigen-positive B cells).

Chimeric antibodies to PSMA were initially isolated having a humanvariable region and a mouse constant region. The antibodies werecharacterized and selected for desirable characteristics, includingaffinity, selectivity, etc. If necessary, mouse constant regions werereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4, to generate a fully human anti-PSMA antibody.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region. The antibody name designations such asH1H11453N2 and H1M11900N denote fully human antibodies “H1H” or chimerichuman variable/mouse constant region antibodies “HIM”. Antibodiesidentified by the hybridoma method are indicated with antibody IDnumbers ending with “N” or “N2”; Antibodies identified by the B-cellsorting method are indicated with antibody ID numbers ending with “P” or“P2”.

Certain biological properties of the exemplary anti-PSMA antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences of Anti-PSMA Antibodies

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-PSMA antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H11453N2 2 4 6 8 16421644 1646 1648 H1H11792P2 10 12 14 16 1642 1644 1646 1648 H1H11797P2 1820 22 24 1642 1644 1646 1648 H1H11800P2 26 28 30 32 1642 1644 1646 1648H1H11803P2 34 36 38 40 1642 1644 1646 1648 H1H11804P2 42 44 46 48 16421644 1646 1648 H1H11805P2 50 52 54 56 1642 1644 1646 1648 H1H11808P2 5860 62 64 1642 1644 1646 1648 H1H11810P2 66 68 70 72 1642 1644 1646 1648H1H11835P2 74 76 78 80 1642 1644 1646 1648 H1H11836P2 82 84 86 88 16421644 1646 1648 H1H11837P2 90 92 94 96 1642 1644 1646 1648 H1H11838P2 98100 102 104 1642 1644 1646 1648 H1H11841P2 106 108 110 112 1642 16441646 1648 H1H11899N2 114 116 118 120 1642 1644 1646 1648 H1H3465P 122124 126 128 130 132 134 136 H1M11900N 138 140 142 144 146 148 150 152

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H11453N2 1 35 7 1641 1643 1645 1647 H1H11792P2 9 11 13 15 1641 1643 1645 1647H1H11797P2 17 19 21 23 1641 1643 1645 1647 H1H11800P2 25 27 29 31 16411643 1645 1647 H1H11803P2 33 35 37 39 1641 1643 1645 1647 H1H11804P2 4143 45 47 1641 1643 1645 1647 H1H11805P2 49 51 53 55 1641 1643 1645 1647H1H11808P2 57 59 61 63 1641 1643 1645 1647 H1H11810P2 65 67 69 71 16411643 1645 1647 H1H11835P2 73 75 77 79 1641 1643 1645 1647 H1H11836P2 8183 85 87 1641 1643 1645 1647 H1H11837P2 89 91 93 95 1641 1643 1645 1647H1H11838P2 97 99 101 103 1641 1643 1645 1647 H1H11841P2 105 107 109 1111641 1643 1645 1647 H1H11899N2 113 115 117 119 1641 1643 1645 1647H1H3465P 121 123 125 127 129 131 133 135 H1M11900N 137 139 141 143 145147 149 151

Example 3: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-PSMA Antibodies

In this example, anti-PSMA antibodies were assessed for their ability tobind to human PSMA. Binding affinities and kinetic constants ofanti-PSMA antibodies to soluble human PSMA protein were determined bysurface plasmon resonance at 37° C. using an antibody-capture format.Results are shown in Tables 3 and 4. Measurements were conducted on aBiacore™ T-200 instrument (GE Healthcare).

Briefly, a CM5 Biacore™ sensor surface was derivatized via aminecoupling with a monoclonal mouse anti-human Fc antibody (GE,#BR-1008-39) or monoclonal goat anti-mouse Fc antibody (GE, #BR-1008-38)to capture purified anti-PSMA antibodies. Binding studies were performedin HBSP++ buffer composed of 0.01M HEPES, 0.15M NaCl, 2 mM Ca²⁺, 2 mMMg²⁺, 0.05% v/v Surfactant P20, pH7.4. Varying concentrations of humanPSMA expressed with an N-terminal hexahistidine tag (6h.hPSMA, R&D)prepared in HBSP++ running buffer (ranging from 50 to 0.78 nM, 4-folddilutions) were injected over the anti-PSMA antibody captured surface ata flow rate of 30 μL/minute. Antibody-reagent association was monitoredfor 2 minutes while dissociation in HBSP++ running buffer was monitoredfor 8 minutes.

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by fitting the real-time sensorgrams to a 1:1 binding modelusing Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t_(1/2) (min)=(In2/(60*k_(d)).

As shown in Tables 3 and 4, the anti-PSMA antibodies of the inventionbound to human PSMA in the antibody capture format with varyingaffinities and K_(D) values ranging from 19.9 pM to 75.6 nM. Severalexemplary anti-PSMA antibodies, such as H1H3465P and H1H11810P2,displayed strong affinity to human PSMA protein, with sub-nanomolarK_(D) values.

TABLE 3 Affinities of anti-PSMA human IgG1 antibodies to soluble humanPSMA at 37° C. Antibody ID ka (1/Ms) kd (1/s) KD (M) t_(1/2) (min)H1H3465P 2.67E+05 6.06E−05 2.27E−10 190.6 H1H11792P2 4.73E+05 9.70E−052.05E−10 119.1 H1H11797P2 1.68E+05 6.30E−04 3.74E−09 18.3 H1H11800P22.51E+05 4.10E−05 1.63E−10 281.8 H1H11803P2 4.57E+05 6.08E−04 1.33E−0919 H1H11804P2 2.03E+04 8.01E−04 3.94E−08 14.4 H1H11805P2 1.29E+059.74E−03 7.56E−08 1.2 H1H11808P2 1.78E+05 ≤1E−05 ≤5.63E−11 ≤1.155H1H11810P2 5.03E+05 ≤1E−05 ≤1.99E−11 ≤1.155 H1H11835P2 1.75E+05 2.46E−031.40E−08 4.7 H1H11836P2 3.27E+05 2.80E−04 8.55E−10 41.3 H1H11837P24.41E+05 7.34E−04 1.66E−09 15.7 H1H11838P2 2.37E+05 4.71E−04 1.99E−0924.5 H1H11841P2 IC IC IC IC IC: inconclusive

TABLE 4 Affinities of anti-PSMA mouse constant antibodies to solublehuman PSMA at 37° C. Antibody ID ka (1/Ms) kd (1/s) KD (M) t_(1/2) (min)H2M11899N 1.51E+05 2.65E−04 1.76E−09 43.6 H1M11453N 1.85E+05 3.56E−041.93E−09 32.4 H1M11900N 1.57E+05 4.06E−03 2.58E−08  2.8

Example 4: Generation of Bispecific Antibodies that BindProstate-Specific Membrane Antigen (PSMA) and CD3

The present invention provides bispecific antigen-binding molecules thatbind CD3 and Prostate-Specific Membrane Antigen (PSMA); such bispecificantigen-binding molecules are also referred to herein as“anti-PSMA/anti-CD3 bispecific molecules.” The anti-PSMA portion of theanti-PSMA/anti-CD3 bispecific molecule is useful for targeting tumorcells that express PSMA, and the anti-CD3 portion of the bispecificmolecule is useful for activating T-cells. The simultaneous binding ofPSMA on a tumor cell and CD3 on a T-cell facilitates directed killing(cell lysis) of the targeted tumor cell by the activated T-cell.

Bispecific antibodies comprising an anti-PSMA-specific binding domainand an anti-CD3-specific binding domain were constructed using standardmethodologies, wherein the anti-PSMA antigen binding domain and theanti-CD3 antigen binding domain each comprise different, distinct HCVRspaired with a common LCVR. In some instances the bispecific antibodieswere constructed utilizing a heavy chain from an anti-CD3 antibody, aheavy chain from an anti-PSMA antibody and a common light chain In otherinstances, the bispecific antibodies were constructed utilizing a heavychain from an anti-CD3 antibody, a heavy chain from an anti-PSMAantibody and a light chain from an anti-CD3 antibody.

The bispecific antibodies described in the following examples consist ofbinding arms known to bind to human soluble heterodimeric hCD3_(ε)/δprotein (as described in Examples 9-13 herein); and human PSMA (seeExamples 1-3 above). Exemplified bispecific antibodies were manufacturedhaving a modified (chimeric) IgG4 Fc domain as set forth in US PatentApplication Publication No. US20140243504A1, published on Aug. 28, 2014.

A summary of the component parts of the antigen-binding domains of thevarious anti-PSMAxCD3 bispecific antibodies constructed is set forth inTable 5.

TABLE 5 Summary of Component Parts of PSMAxCD3 Bispecific AntibodiesAnti-PSMA Anti-CD3 Antigen-Binding Antigen-Binding Common Domain DomainLight Chain Bispecific Heavy Chain Heavy Chain Variable AntibodyIdentifier Variable Region Variable Region Region BSPSMA/CD3-001PSMA-VH-3465 CD3-VH-A CD3-VL-A BSPSMA/CD3-002 PSMA-VH-3465 CD3-VH-BCD3-VL-B BSPSMA/CD3-003 PSMA-VH-11810 CD3-VH-G VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-200 PSMA-VH-11810 CD3-VH-G2 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-300 PSMA-VH-11810 CD3-VH-G3 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-400 PSMA-VH-11810 CD3-VH-G4 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-004 PSMA-VH-11810 CD3-VH-G5 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-800 PSMA-VH-11810 CD3-VH-G8 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3-900 PSMA-VH-11810 CD3-VH-G9 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G10 VK 1-39 JK 5 1000 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G11 VK 1-39 JK 5 1100 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G12 VK 1-39 JK 5 1200 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G13 VK 1-39 JK 5 1300 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G14 VK 1-39 JK 5 1400 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G15 VK 1-39 JK 5 1500 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G16 VK 1-39 JK 5 1600 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G17 VK 1-39 JK 5 1700 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G18 VK 1-39 JK 5 1800 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G19 VK 1-39 JK 5 1900 (SEQ ID NO:1386) BSPSMA/CD3-005 PSMA-VH-11810 CD3-VH-G20 VK 1-39 JK 5 (SEQ ID NO:1386) BSPSMA/CD3- PSMA-VH-11810 CD3-VH-G21 VK 1-39 JK 5 2100 (SEQ ID NO:1386)

The anti-PSMA heavy chain variable region PSMA-VH-3465 is the HCVR ofH1H3465P (SEQ ID NO:122) from Table 1. The anti-PSMA heavy chainvariable region PSMA-VH-11810 is the HCVR of H1H11810P2 (SEQ ID NO:66)from Table 1.

The anti-CD3 heavy chain variable region CD3-VH-A is the HCVR ofH1H5778P (SEQ ID NO:922) from Table 12. The anti-CD3 heavy chainvariable region CD3-VH-B is the HCVR of H1H2712N (SEQ ID NO:154) fromTable 12. The anti-CD3 heavy chain variable regions CD3-VH-G, CD3-VH-G2,CD3-VH-G3, CD3-VH-G4, CD3-VH-G5, CD3-VH-G8, CD3-VH-G9, CD3-VH-G10,CD3-VH-G11, CD3-VH-G12, CD3-VH-G13, CD3-VH-G14, CD3-VH-G15, CD3-VH-G16,CD3-VH-G17, CD3-VH-G18, CD3-VH-G19, CD3-VH-G20, and CD3-VH-G21 aredescribed in Table 18.

The light chains in Table 5 were common to both the CD3 and PSMAtargeting arms of the bispecific antibodies. The anti-CD3 light chainvariable region CD3-VL-A is the LCVR of H1H5778P (SEQ ID NO:930) fromTable 12. The anti-CD3 light chain variable region CD3-V_(L)-B is theLCVR of H1H2712N (SEQ ID NO:162) from Table 12. The light chain variableregion VK 1-39 JK 5 is SEQ ID NO: 1386 from Table 20. Table 1 sets outamino acid sequence identifiers for the various heavy chain variableregions, and their corresponding CDRs, of the anti-PSMA arms of thebispecific antibodies of this Example. Table 2 sets out the sequenceidentifiers for the nucleotide sequences encoding the heavy chainvariable regions, and their corresponding CDRs, of the anti-PSMAantigen-binding domains of the bispecific antibodies of this Example.

Tables 12, 14, 15, 18, and 20 describe amino acid sequences of the heavychain variable regions, and their corresponding CDRs, for the anti-CD3arms of the bispecific antibodies, as well as amino acid sequences forthe light chain variable regions, and their corresponding CDRs, commonto both arms of the bispecific antibodies of this Example. Tables 13,16, 17, 19, and 21 describe the corresponding nucleotide sequences forthese features of the bispecific antibodies of this Example.

Example 5: Binding Affinities of Exemplified Bispecific Antibodies asMeasured by FACS Analysis

In this example, the ability of the anti-PSMA/anti-CD3 bispecificantibodies described in Example 4 to bind to human PSMA expressing celllines and to human and cynomolgus CD3-expressing cell lines via FACS wasdetermined. As described above, the bispecific antibodies of thisinvention utilized a PSMA-specific heavy chain (HC) binding arm pairedwith a panel of anti-CD3 HC binding arms and a common light chain. ThePSMA-HC binding arms in the bispecific antibodies, below, demonstratedpotent binding to human PSMA protein via surface plasmon resonance(Example 3). As described in Examples 6 and 13 herein, the CD3-bindingHC arms also displayed a range of affinities to human solubleheterodimeric hCD3_(ε)/δ.mFc protein via surface plasmon resonance.

Briefly, 2×10⁵ cells/well of human CD3-expressing Jurkat, cynomolgus T,or human PSMA-specific expressing cells were incubated with a serialdilution of bispecific antibodies for 30 min at 4° C. After incubation,cells were washed and a goat F(ab′)₂ anti-human Fcγ PE labeled secondary(Jackson Immunolabs) was added to the cells for an additional 30 min.Next, cells were washed, re-suspended in cold PBS+1% BSA and analyzedvia flow cytometry on a BD FACS Canto II.

For FACS analysis, cells were gated by forward scatter height vs.forward scatter area for single events selection, followed by side andforward scatters. The EC₅₀ for cell binding titration was determinedusing Prism software. Values were calculated using 4-parameternon-linear regression analysis.

TABLE 6 FACS Binding on CD3 and PSMA-Specific Cell lines BispecificAnti-CD3- Cyno T- B16F10.9/ Antibody Binding Jurkat cells PSMA 22RV1Identifier Arm EC₅₀ [M] EC₅₀ [M] EC₅₀ [M] EC₅₀ [M] BSPSMA/ CD3-VH-3.91E−08 NT NT 7.85E−08 CD3-001 A BSPSMA/ CD3-VH- NT NT NT NT CD3-002 BBSPSMA/ CD3-VH- 1.65E−08 1.42E−08 2.26E−09 NT CD3-003 G BSPSMA/ CD3-VH-NB NB 1.88E−09 NT CD3-200 G2 BSPSMA/ CD3-VH- NB NB 1.90E−09 NT CD3-300G3 BSPSMA/ CD3-VH- NB NB 1.72E−09 NT CD3-400 G4 BSPSMA/ CD3-VH- ~1.0E−06NB 1.31E−09 NT CD3-004 G5 BSPSMA/ CD3-VH- 1.93E−08 1.96E−08 1.31E−09 NTCD3-800 G8 BSPSMA/ CD3-VH- 2.74E−07 NB 1.43E−09 NT CD3-900 G9 BSPSMA/CD3-VH- 2.77E−07 NB 1.19E−09 NT CD3-1000 G10 BSPSMA/ CD3-VH- 1.83E−088.90E−07 1.03E−09 NT CD3-1100 G11 BSPSMA/ CD3-VH- 4.72E−08 NB 1.16E−09NT CD3-1200 G12 BSPSMA/ CD3-VH- 1.02E−07 2.17E−06 1.25E−09 NT CD3-1300G13 BSPSMA/ CD3-VH- 3.19E−08 1.70E−07 1.30E−09 NT CD3-1400 G14 BSPSMA/CD3-VH- 9.30E−08 NB 1.21E−09 NT CD3-1500 G15 BSPSMA/ CD3-VH- 5.68E−08 NB1.03E−09 NT CD3-1600 G16 BSPSMA/ CD3-VH- 2.00E−07 3.35E−06 1.34E−09 NTCD3-1700 G17 BSPSMA/ CD3-VH- 1.26E−07 NB 2.16E−09 NT CD3-1800 G18BSPSMA/ CD3-VH- 6.07E−08 NB 1.35E−09 NT CD3-1900 G19 BSPSMA/ CD3-VH-2.10E−07 6.14E−06 2.09E−09 NT CD3-005 G20 BSPSMA/ CD3-VH- 1.06E−07 NB1.14E−09 NT CD3-2100 G21 NB = no binding; NT = not tested

As shown in Table 6, the anti-PSMA/anti-CD3 bispecific antibodies testeddemonstrated specificity of binding to human PSMA-expressingB16F10.9/hPSMA and 22RV1 cell lines via FACS. The detection limit forFACS binding is 1 pM EC50.

As shown in Table 6, the CD3 binding arms of each CD3×PSMA bispecificantibody displayed a range of cell binding affinity to human CD3expressing Jurkat cells (15 to 300 nM EC50 range). Importantly, the CD3arms that showed weak-to-no binding to human CD3 heterodimeric proteinvia surface plasmon resonance (see Table 7 hereinbelow) also correlatedwith weak to no observable binding on Jurkat cells (i.e. CD3-VH-G2,CD3-VH-G3, CD3-VH-G5). Several CD3-binding arms also displayed crossreactivity to cynomolgus T-cells. All tested bispecific antibodiesdisplayed similar cell binding on respective PSMA-expressing cell lines,confirming that bispecific pairing with individual CD3 arms did notaffect or diminish PSMA-specific binding (PSMA-specific binding was lessthan or equal to 5.6 nM (high affinity) in all examples tested).

Antibodies exhibiting weak-to-no detectable binding to human CD3, andalso exhibiting weak-to-no binding to cynomolgus CD3, were consideredadvantageous for avidity-driven bispecific pairing in accordance withthe present invention, and were further tested for cytotoxicity in invitro and in vivo assays.

Example 6: Binding Affinities of Exemplified Antibodies as Measured by aSurface Plasmon Resonance Binding Assay

Binding affinities and kinetic constants of anti-PSMA× anti-CD3bispecific antibodies to soluble heterodimeric hCD3_(ε)/δ.mFc protein(hCD3ε=UniProtKB/Swiss-Prot: P07766.2; SEQ_ID NO: 1652;hCD3δ=UniProtKB/Swiss-Prot: P04234.1, SEQ ID NO: 1653) were determinedby surface plasmon resonance at 37° C. using an antigen-capture format(Table 7). Measurements were conducted on a Sierra Sensors MASS-1instrument.

In the antigen-capture format, the MASS-1 high-density amine sensorsurface was derivatized with a goat anti-mouse IgG2a polyclonal antibody(Southern Biotech). Soluble heterodimeric CD3 protein was captured andthe respective antibodies were injected over the captured antigen.

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing MASS-1 AnalyserR2 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t112) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t112 (min)=(In2/(60*k_(d)).

TABLE 7 Affinities of anti-CD3 Bispecific Antibodies to Soluble HumanCD3 Binding at 37° C./Antigen-Capture Format Corresponding anti-Bispecific CD3 Antigen- Antibody Binding HCVR T½ Identifier Identifierka (Ms⁻¹) kd (s⁻¹) K_(D) (M) (min) BSPSMA/CD3- CD3-VH-G 1.32E+057.62E−04 5.78E−09 15.2 003 BSPSMA/CD3- CD3-VH-G2 NB NB NB NB 200BSPSMA/CD3- CD3-VH-G3 NB NB NB NB 300 BSPSMA/CD3- CD3-VH-G4 NB NB NB NB400 BSPSMA/CD3- CD3-VH-G5 NB NB NB NB 004 BSPSMA/CD3- CD3-VH-G8 5.95E+041.15E−03 1.94E−08 10.0 800 BSPSMA/CD3- CD3-VH-G9 4.38E+04 4.95E−031.13E−07 2.3 900 BSPSMA/CD3- CD3-VH-G10 3.44E+04 6.37E−03 1.85E−07 1.81000 BSPSMA/CD3- CD3-VH-G11 9.21E+04 1.02E−03 1.11E−08 11.3 1100BSPSMA/CD3- CD3-VH-G12 3.85E+04 2.47E−03 6.42E−08 4.7 1200 BSPSMA/CD3-CD3-VH-G13 2.03E+04 2.48E−03 1.22E−07 4.7 1300 BSPSMA/CD3- CD3-VH-G146.21E+04 3.31E−03 5.33E−08 3.5 1400 BSPSMA/CD3- CD3-VH-G15 7.36E+046.11E−03 8.29E−08 1.9 1500 BSPSMA/CD3- CD3-VH-G16 6.43E+04 2.43E−033.78E−08 4.7 1600 BSPSMA/CD3- CD3-VH-G17 4.70E+04 3.07E−03 6.52E−08 3.81700 BSPSMA/CD3- CD3-VH-G18 NB NB NB NB 1800 BSPSMA/CD3- CD3-VH-G194.43E+04 5.09E−03 1.15E−07 2.3 1900 BSPSMA/CD3- CD3-VH-G20 1.73E+045.77E−03 3.34E−07 2.0 005 BSPSMA/CD3- CD3-VH-G21 3.02E+04 2.34E−037.75E−08 4.9 2100 Control 1 CD3−L2K 3.68E+05 2.66E−03 7.22E−09 4.3

As shown in Table 7, the anti-CD3×anti-PSMA bispecific antibodies eithermaintained very weak binding to soluble CD3 in the surface plasmonresonance binding assay, e.g. having a K_(D) value greater than 11 nM upto 334 nM which is weaker than that of the bispecific anti-CD3 armderived from germline frameworks, CD3-V_(H)-G, or did not exhibit anydetectable binding.

As such, several bispecific antibodies exhibited greater than 50 nM KDvalues, and some were greater than 100 nM (i.e. BSPSMA/CD3-900,BSPSMA/CD3-1000, BSPSMA/CD3-1900, BSPSMA/CD3-005) and even beyond thedetection limit of the assay, i.e. showed no detectable binding tosoluble human CD3 (i.e. BSPSMA/CD3-200, BSPSMA/CD3-300, BSPSMA/CD3-400,BSPSMA/CD3-004 and BSPSMA/CD3-1800).

Example 7: T Cell Activation and Tumor-Specific Cytotoxicity Exhibitedby Bispecific Antibodies of the Invention as Measured In Vitro

In this example, the specific killing of PSMA-expressing target cells inthe presence of anti-PSMA×anti-CD3 bispecific antibodies was monitoredvia flow cytometry. As reported previously, the bispecific antibodiesdisplayed a range of affinity to CD3 protein and CD3-expressing celllines (i.e. weak, moderate and strong binding). This same panel ofbispecific antibodies was tested for the ability to induce naïve humanT-cells to re-direct killing toward target-expressing cells.

Briefly, PSMA-expressing (C4-2, 22Rv1 and TRAMPC2_PSMA) cell lines werelabeled with 1 μM of the fluorescent tracking dye Violet Cell Tracker.After labeling, cells were plated overnight at 37° C. Separately, humanPBMCs were plated in supplemented RPMI media at 1×10⁶ cells/mL andincubated overnight at 37° C. in order to enrich for lymphocytes bydepleting adherent macrophages, dendritic cells, and some monocytes. Thenext day, target cells were co-incubated with adherent cell-depletednaïve PBMC (Effector/Target cell 4:1 ratio) and a serial dilution ofrelevant bispecific antibodies or Isotype control (concentration range:66.7 nM to 0.25 pM) for 48 hours at 37° C. Cells were removed from cellculture plates using an enzyme-free cell dissociation buffer, andanalyzed by FACS.

For FACS analysis, cells were stained with a dead/live far red celltracker (Invitrogen). 5×10⁵ counting beads were added to each wellimmediately before FACS analysis. 1×10⁴ beads were collected for eachsample. For the assessment of specificity of killing, cells were gatedon live Violet labeled populations. Percent of live population wasrecorded and used for the calculation of normalized survival.

T cell activation was assessed by incubating cells with directlyconjugated antibodies to CD2 and CD69, and by reporting the percent ofactivated (CD69+) T cells out of total T cells (CD2+).

As the results in Table 8 show, depletion of PSMA-expressing cells wasobserved with anti-PSMA×anti-CD3 bispecific antibodies. Most of thetested bispecific antibodies activated and directed human T cells todeplete the target cells with EC₅₀s in picomolar range. Additionally,the observed target-cell lysis was associated with an up-regulation ofCD69 cells on CD2+ T cells, with pM EC₅₀s.

Importantly, the results of this example demonstrate that severalbispecifics which utilized a CD3 binding arm that displayedweak-to-non-observable binding to CD3 protein or CD3-expressing cells(i.e. CD3-VH-G5) still retained the ability to activate T-cells andexhibited potent cytotoxicity of tumor antigen-expressing cells.

TABLE 8 Cytotoxicity and T-cell activation properties of selectedPSMAxCD3 Bispecific Antibodies 22RV1 TrampC2. Bispecific Anti-CD3 C4-2Cell Cell PSMA T cell Antibody Binding depletion depletion Celldepletion activation Identifier Arm EC₅₀ [M] EC₅₀ [M] EC₅₀ [M] EC₅₀ [M]BSPSMA/ CD3-VH- 1.03E−11 NT 6.43E−12 1.23E−12 CD3-003 G BSPSMA/ CD3-VH-NT No NT No CD3-200 G2 activity activity BSPSMA/ CD3-VH- NT Very NT1.85E-11 CD3-300 G3 weak BSPSMA/ CD3-VH- NT Very NT Very CD3-400 G4 weakweak BSPSMA/ CD3-VH- 2.15E−11 6.31E−12 1.15E−11 1.34E−11 CD3-004 G5BSPSMA/ CD3-VH- NT NT 9.27E−12 1.76E−12 CD3-800 G8 BSPSMA/ CD3-VH- NT NT3.50E−12 1.12E−12 CD3-900 G9 BSPSMA/ CD3-VH- NT NT 5.97E−12 1.28E−12CD3-1000 G10 BSPSMA/ CD3-VH- NT NT 3.86E−12 1.11E−12 CD3-1100 G11BSPSMA/ CD3-VH- 8.74E-12 NT NT 2.31E−12 CD3-1300 G13 BSPSMA/ CD3-VH-7.37E-12 2.07E−12 NT 3.89E−12 CD3-1700 G17 BSPSMA/ CD3-VH- 1.39E-118.32E−12 NT 6.11E−12 1 CD3-005 G20 NT = not tested

Example 8: Anti-PSMA/Anti-CD3 Bispecific Antibodies Display PotentAnti-Tumor Efficacy In Vivo

To determine the in vivo efficacy of exemplary anti-PSMA/anti-CD3bispecific antibodies, studies were performed in immunocompromised micebearing human prostate cancer xenografts. Additional studies were alsocarried out in immunocompetent mice bearing mouse prostate cancerxenografts engineered to express human PSMA.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies in Human TumorXenograft Models

To assess the in vivo efficacy of the anti-PSMA/anti-CD3 bispecifics inhuman tumor xenograft studies, NOD scid gamma (NSG) mice (JacksonLaboratories, Bar Harbor, Me.) were co-implanted with human peripheralblood mononuclear cells (PBMCs) along with 22Rv1 or C4-2 human prostatetumor cells which endogenously express PSMA.

Briefly, 4×10⁶ 22 Rv1 or 5×10⁶ C4-2 cells (MD Anderson, Tex.) cells wereco-implanted s.c. with 1×10⁶ human PBMCs (ReachBio, LLC., Seattle,Wash.) in a 50:50 mix of matrigel matrix (BD Biosciences) into the rightflank of male NSG mice. In the 22Rv1 study, mice were treated i.p. ondays 0, 3 and 7 with 1 ug of BSPSMA/CD3-001 or an isotype control (FIG.1). In the C4-2 study, mice were treated i.p. on days 0, 4, and 7 posttumor implantation with 0.1 mg/kg BSPSMA/CD3-001, BSPSMA/CD3-003 orBSPSMA/CD3-005.

In an additional xenogenic model, anti-PSMA/anti-CD3 bispecifics weretested in mice engrafted with human hematopoietic CD34+ stem cells.Briefly, newborn SIRPα BALB/c-Rag2-IL2rγ-(BRG) pups were engrafted withhCD34+ fetal liver cells. 3-6 months later hCD34-engrafted SIRPa BRGmice were then implanted with C4-2 cells (5×10⁶ s.c. in matrigel). 8days later, mice were treated with 10 ug of BSPSMA/CD3-004 or an isotypecontrol antibody, followed by 2×/week doses throughout the study.

In all studies, tumor size was measured 2×/week using calipers and tumorvolume calculated as Volume=(length×width²)².

As the results in Table 9 show, the bispecific antibodies tested in thexenogenic models described above were all effective at inhibiting tumorgrowth compared to treatment with the isotype control.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies in EstablishedHuman Tumor Xenograft Model

Next, the efficacy of anti-PSMA/anti-CD3 bispecific antibodies insuppressing the growth of established tumors was assessed. NSG mice werefirst injected with 2.5×10⁶ human PBMCs i.p. to allow for engraftment ofhuman T cells. Fourteen days later, mice were co-implanted with C4-2cells and PBMCs as above. 20 ug of BSPSMA/CD3-002 or an isotype controlwere administered i.p. 18 days post tumor implantation and continued2×/week for the duration of the study. Additional PBMCs were given i.p.on days 20 and 40 post tumor implantation.

As the results in Table 9 show, BSPSMA/CD3-002 showed efficacy insuppressing the growth of established tumors, decreasing tumor growth by95%.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies in Immune-CompetentTumor Model

Additionally, anti-PSMA/anti-CD3 bispecifics were assessed foranti-tumor activity in an immune-competent model. Mice humanized for thethree chains (δγε) of CD3 as well as for PSMA were implanted with avariant murine prostate cancer cell line TRAMP-C2 transfected with humanPSMA.

Prior to study initiation, the tumorigenic cell line variantTRAMP-02_hPSMAv #1 was generated. Briefly, 7.5×10⁶ TRAMP-C2_hPSMA cellswere implanted s.c. into the right flank of male mice humanized for CD3and PSMA. A tumor was excised and cut into 3 mm fragments andsubsequently implanted into the right flank of new male humanized mice.A tumor arising from the implanted tumor fragments was then harvestedand disaggregated into a single cell suspension. These cells(TRAMP-C2_hPSMAv #1) were then cultured in vitro under G418 selection.4.10⁶ cells of this variant cell line were then implanted into the rightflank of male PSMA/CD3 humanized mice for the bispecific antibodyefficacy studies.

Humanized PSMA/CD3 mice implanted with TRAMPC2_hPSMAv #1 were treatedwith 100 ug or 10 ug of anti-PSMA/anti-CD3 bispecific antibodyBSPSMA/CD3-001 or BSPSMA/CD3-004 or an isotype control 2×/week startingfrom the day of tumor implantation. Serum cytokine levels 4hpost-injection were also examined, as well as spleen T-cell levels.Study was terminated at Day 27.

As the results in Table 10 show, both anti-PSMA/anti-CD3 bispecificmolecules showed efficacy in significantly delaying tumor growth acrosstreatment groups. Dose dependent cytokine release was observed aftertreatment with BSPSMA/CD3-001. Minimal cytokine release was observedafter administration of BSPSMA/CD3-004, possibly due to the weak bindingof the anti-CD3. BSPSMA/CD3-001 showed anti-tumor efficacy withoutdepleting T cells in the spleen.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies on EstablishedTumors in Immune-Competent Model

Lastly, the efficacy of selected anti-PSMA/anti-CD3 bispecific moleculeson reducing growth of established tumors in humanized PSMA/CD3 mice wasassessed. TRAMP-C2_hPSMAv #1 cells were transplanted in humanized miceas described above, and 100 ug BSPSMA/CD3-001 or isotype control wasadministered i.p. 2×/week 14 days after tumor implantation, when tumorsizes ranged from 50 mm³-100 mm³. As the results in Table 11 show,BSPSMA/CD3-001 was efficacious in this established tumor model,displaying an 84% decrease in tumor growth compared to the controlgroup.

In summary, the anti-PSMA/anti-CD3 bispecific antibodies of thisinvention display potent anti-tumor efficacy in both immune-compromisedand immune-competent tumor models. Additionally, several of the testedbispecific antibodies (BSPSMA/CD3-001 and 002) displayed potent abilityto reduce the volume of established tumors.

Of note, in the absence of PSMA-expressing tumor cells, no T cellactivation was seen.

Additionally, in mice bearing no tumors, blood samples were collected 4hours following PSMAxCD3 bispecific antibody treatment, and serumcytokine levels were determined. Transient increases in levels ofcytokines, namely interferon-gamma (IFN-g), tumor necrosis factor (TNF),interleukin-2 (IL-2), and interleukin-6 (IL-6) were determined and thetransient increases were dose-dependent (FIGS. 2A-2D).

In order to validate the specificity of the bispecific antibodies, miceeither humanized for both PSMA and CD3 or mice humanized for CD3 alonewere dosed with 100 μg of PSMAxCD3 and examined for serum cytokines (4hrs post dose) and transient T cell loss from the blood (24 hrs postdose). Treatment with PSMAxCD3 bispecific antibody in a humanized T cellmouse (100 μg/mouse) induces acute increase in cytokines (e.g. IFNg) aswell as transient decrease in circulating T cells (FIGS. 3A-3B). Thisfinding reproduces cytokine and T cell changes that have been observedin human patients treated with tumor antigen×CD3 bispecific antibodies.

PSMAxCD3 bispecific antibodies are efficacious without depletingeffector T cells in the spleen of the immunocompetent mice, as shown inFIGS. 4A-4C. Briefly, humanized PSMA and CD3 Velocigene® mice wereimplanted with hPSMA-expressing tumors and treated with PSMAxCD3 twiceweekly. T cells were present at normal numbers at final harvest. Spleenswere examined for CD4+ T cells, CD8+ T cells, and Tregs at the end ofthe experiment after treatment with PSMAxCD3 bispecific antibody twiceper week throughout study. Mice humanized for PSMA and CD3 wereimplanted with TRAMP-C2_hPSMA tumors and dosed from day 0 with 100 μg or10 μg of PSMAxCD3. Cell populations in the spleen were analyzed by flowcytometry. Data was analyzed using analysis of variance (ANOVA) for anysignificant effects compared to the isotype control group but nosignificant differences were found (FIGS. 4A-4C).

TABLE 9 Efficacy of anti-PSMA/anti-CD3 Bispecific Antibodies inImmune-Compromised Xenograft Models Xenogenic model: suppression oftumor growth N Final Tumor Volume Tumor Model/ #mice/ BispecificAntibody (mm³) Mouse Strain treatment group Identifier Dose Mean ± SD22Rv1/ 5 BSPSMA/CD3-001 1.0 ug/mouse 370 ± 270 NSG 5 Isotype Control onday 0, 3 & 7 1260 ± 730  C4-/2 5 BSPSMA/CD3-001 0.1 mg/kg 0 ± 0 NSG 5BSPSMA/CD3-003 on day 0, 4 & 7 0 ± 0 5 BSPSMA/CD3-005 0 ± 0 5 IsotypeControl 960 ± 660 C4-2/ 5 BSPSMA/CD3-004 1.0 ug/mouse 70 ± 60 SIRPαBalb/c-Rag2- 2×/week IL2rγ- BRG engrafted with hCD34+ HSC 5 IsotypeControl 260 ± 180 Xenogenic model: inhibition of established tumorgrowth Tumor Growth N Bispecific Antibody (mm³) from start of TumorModel/ # mice/ Identifier treatment % Decrease Tumor Mouse Straintreatment group Dose: 20 ug/ mouse (mean ± SD) Growth vs. Control C4-2/5 BSPSMA/CD3-002  60 ± 100 95% NSG 4 Isotype Control 1170 ± 600  (−)

TABLE 10 Efficacy of anti-PSMA/anti-CD3 Bispecific antibodies inimmune-competent syngeneic models N Tumor Spleen T-cell Dose # mice/Volume (mm³) Mean Serum Cytokine level %, Tumor Model/ BispecificAntibody (ug/mouse) treatment at Day 27 Concentrations, (pg/mL) (mean ±SD)^(#) Mouse Strain Identifier 2x/week* group (Mean ± SD) IFNg TNFaIL-2 IL-12p70 IL-6 CD4+ CD8+ TRAMP-C2/ BSPSMA/CD3-001 100 4  260 ± 250130 180 150 70 20 6.0 ± 1.0 13.0 ± 3.0 PSMA^(Hum/hum) 10 6  600 ± 330800 140 140 30 1130 7.0 ± 2.0 14.0 ± 3.0 CD3^(Hum/Hum) BSPSMA/CD3-004100 4  50 ± 60 30 60 60 40 370 8.0 ± 1.0 12.0 ± 2.0 10 5  380 ± 650 1050 50 10 330 8.0 ± 3.0 14.0 ± 4.0 Isotype Control 100 5 1740 ± 560 4 3030 10 230 5.0 ± 1.0  8.0 ± 2.0 *Mice were dosed with antibody or isotypecontrol 2x/week starting on the day of tumor implantation ^(#)Measuredas the percentage of CD4+ or CD8+ cells in spleen out of live mCD45+cells

TABLE 11 Efficacy of anti-PSMA/anti-CD3 Bispecific antibodies insuppression of established tumor growth in immune competent syngeneicmodel Tumor efficacy in immune-competent model, established tumors TumorGrowth (mm³) from Tumor Model/ Bispecific Antibody Identifier start oftreatment % Decrease Tumor Mouse Strain Dose: 100 ug/mouse (mean ± SD)Growth vs. Control TRAMP-C2/ BSPSMA/CD3-001 170 ± 170 84 psmA ^(Hum/hum)Isotype Control 740 ± 570 (−) CD3 ^(Hum/Hum)

Example 9: Generation of Anti-CD3 Antibodies

Anti-CD3 antibodies were obtained by immunizing an engineered mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions with cells expressing CD3 or with DNA encoding CD3. Theantibody immune response was monitored by a CD3-specific immunoassay.When a desired immune response was achieved, splenocytes were harvestedand fused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce CD3-specific antibodies.Using this technique several anti-CD3 chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained. In addition, several fully human anti-CD3 antibodies wereisolated directly from antigen-positive B cells without fusion tomyeloma cells, as described in US 2007/0280945A1.

Certain biological properties of the exemplary anti-CD3 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples herein.

Example 10: Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 12 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-CD3 antibodies ofthe invention. The corresponding nucleic acid sequence identifiers areset forth in Table 13. Methods of making the anti-CD3 antibodiesdisclosed herein can also be found in US publication 2014/0088295.

TABLE 12 Amino Acid Sequence Indentifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H2712N 154 156158 160 162 164 166 168 H1M2692N 170 172 174 176 178 180 182 184H1M3542N 186 188 190 192 194 196 198 200 H1M3544N 202 204 206 208 210212 214 216 H1M3549N 218 220 222 224 226 228 230 232 H1M3613N 234 236238 240 242 244 246 248 H2M2689N 250 252 254 256 258 260 262 264H2M2690N 266 268 270 272 274 276 278 280 H2M2691N 282 284 286 288 290292 294 296 H2M2704N 298 300 302 304 306 308 310 312 H2M2705N 314 316318 320 322 324 326 328 H2M2706N 330 332 334 336 338 340 342 344H2M2707N 346 348 350 352 354 356 358 360 H2M2708N 362 364 366 368 370372 374 376 H2M2709N 378 380 382 384 386 388 390 392 H2M2710N 394 396398 400 402 404 406 408 H2M2711N 410 412 414 416 418 420 422 424H2M2774N 426 428 430 432 434 436 438 440 H2M2775N 442 444 446 448 450452 454 456 H2M2776N 458 460 462 464 466 468 470 472 H2M2777N 474 476478 480 482 484 486 488 H2M2778N 490 492 494 496 498 500 502 504H2M2779N 506 508 510 512 514 516 518 520 H2M2789N 522 524 526 528 530532 534 536 H2M2862N 538 540 542 544 546 548 550 552 H2M2885N 554 556558 560 562 564 566 568 H2M2886N 570 572 574 576 578 580 582 584H2M3540N 586 588 590 592 594 596 598 600 H2M3541N 602 604 606 608 610612 614 616 H2M3543N 618 620 622 624 626 628 630 632 H2M3547N 634 636638 640 642 644 646 648 H2M3548N 650 652 654 656 658 660 662 664H2M3563N 666 668 670 672 674 676 678 680 H1H5751P 682 684 686 688 690692 694 696 H1H5752P 698 700 702 704 706 708 710 712 H1H5753B 714 716718 720 722 724 726 728 H1H5754B 730 732 734 736 738 740 742 744H1H5755B 746 748 750 752 754 756 758 760 H1H5756B 762 764 766 768 770772 774 776 H1H5757B 778 780 782 784 786 788 790 792 H1H5758B 794 796798 800 802 804 806 808 H1H5761P 810 812 814 816 818 820 822 824H1H5763P 826 828 830 832 834 836 838 840 H1H5764P 842 844 846 848 850852 854 856 H1H5769P 858 860 862 864 866 868 870 872 H1H5771P 874 876878 880 882 884 886 888 H1H5772P 890 892 894 896 898 900 902 904H1H5777P 906 908 910 912 914 916 918 920 H1H5778P 922 924 926 928 930932 934 936 H1H5780P 938 940 942 944 946 948 950 952 H1H5781P 954 956958 960 962 964 966 968 H1H5782P 970 972 974 976 978 980 982 984H1H5785B 986 988 990 992 994 996 998 1000 H1H5786B 1002 1004 1006 10081010 1012 1014 1016 H1H5788P 1018 1020 1022 1024 1026 1028 1030 1032H1H5790B 1034 1036 1038 1040 1042 1044 1046 1048 H1H5791B 1050 1052 10541056 1058 1060 1062 1064 H1H5792B 1066 1068 1070 1072 1074 1076 10781080 H1H5793B 1082 1084 1086 1088 1090 1092 1094 1096 H1H5795B 1098 11001102 1104 1106 1108 1110 1112 H1H5796B 1114 1116 1118 1120 1122 11241126 1128 H1H5797B 1130 1132 1134 1136 1138 1140 1142 1144 H1H5798B 11461148 1150 1152 1154 1156 1158 1160 H1H5799P 1162 1164 1166 1168 11701172 1174 1176 H1H5801B 1178 1180 1182 1184 1186 1188 1190 1192 H1H7194B1194 1196 1198 1200 1386 1388 1390 1392 H1H7195B 1202 1204 1206 12081386 1388 1390 1392 H1H7196B 1210 1212 1214 1216 1386 1388 1390 1392H1H7198B 1218 1220 1222 1224 1386 1388 1390 1392 H1H7203B 1226 1228 12301232 1386 1388 1390 1392 H1H7204B 1234 1236 1238 1240 1386 1388 13901392 H1H7208B 1242 1244 1246 1248 1386 1388 1390 1392 H1H7211B 1250 12521254 1256 1386 1388 1390 1392 H1H7221B 1258 1260 1262 1264 1386 13881390 1392 H1H7223B 1266 1268 1270 1272 1386 1388 1390 1392 H1H7226B 12741276 1278 1280 1386 1388 1390 1392 H1H7232B 1282 1284 1286 1288 13861388 1390 1392 H1H7233B 1290 1292 1294 1296 1386 1388 1390 1392 H1H7241B1298 1300 1302 1304 1386 1388 1390 1392 H1H7242B 1306 1308 1310 13121386 1388 1390 1392 H1H7250B 1314 1316 1318 1320 1386 1388 1390 1392H1H7251B 1322 1324 1326 1328 1386 1388 1390 1392 H1H7254B 1330 1332 13341336 1386 1388 1390 1392 H1H7258B 1338 1340 1342 1344 1386 1388 13901392 H1H7269B 1346 1348 1350 1352 1386 1388 1390 1392 H1H7279B 1354 13561358 1360 1386 1388 1390 1392 H1xH7221G 1362 1364 1366 1368 1386 13881390 1392 H1xH7221G3 1370 1372 1374 1376 1386 1388 1390 1392 H1xH7221G51378 1380 1382 1384 1386 1388 1390 1392

TABLE 13 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H2712N 153155 157 159 161 163 165 167 H1M2692N 169 171 173 175 177 179 181 183H1M3542N 185 187 189 191 193 195 197 199 H1M3544N 201 203 205 207 209211 213 215 H1M3549N 217 219 221 223 225 227 229 231 H1M3613N 233 235237 239 241 243 245 247 H2M2689N 249 251 253 255 257 259 261 263H2M2690N 265 267 269 271 273 275 277 279 H2M2691N 281 283 285 287 289291 293 295 H2M2704N 297 299 301 303 305 307 309 311 H2M2705N 313 315317 319 321 323 325 327 H2M2706N 329 331 333 335 337 339 341 343H2M2707N 345 347 349 351 353 355 357 359 H2M2708N 361 363 365 367 369371 373 375 H2M2709N 377 379 381 383 385 387 389 391 H2M2710N 393 395397 399 401 403 405 407 H2M2711N 409 411 413 415 417 419 421 423H2M2774N 425 427 429 431 433 435 437 439 H2M2775N 441 443 445 447 449451 453 455 H2M2776N 457 459 461 463 465 467 469 471 H2M2777N 473 475477 479 481 483 485 487 H2M2778N 489 491 493 495 497 499 501 503H2M2779N 505 507 509 511 513 515 517 519 H2M2789N 521 523 525 527 529531 533 535 H2M2862N 537 539 541 543 545 547 549 551 H2M2885N 553 555557 559 561 563 565 567 H2M2886N 569 571 573 575 577 579 581 583H2M3540N 585 587 589 591 593 595 597 599 H2M3541N 601 603 605 607 609611 613 615 H2M3543N 617 619 621 623 625 627 629 631 H2M3547N 633 635637 639 641 643 645 647 H2M3548N 649 651 653 655 657 659 661 663H2M3563N 665 667 669 671 673 675 677 679 H1H5751P 681 683 685 687 689691 693 695 H1H5752P 697 699 701 703 705 707 709 711 H1H5753B 713 715717 719 721 723 725 727 H1H5754B 729 731 733 735 737 739 741 743H1H5755B 745 747 749 751 753 755 757 759 H1H5756B 761 763 765 767 769771 773 775 H1H5757B 777 779 781 783 785 787 789 791 H1H5758B 793 795797 799 801 803 805 807 H1H5761P 809 811 813 815 817 819 821 823H1H5763P 825 827 829 831 833 835 837 839 H1H5764P 841 843 845 847 849851 853 855 H1H5769P 857 859 861 863 865 867 869 871 H1H5771P 873 875877 879 881 883 885 887 H1H5772P 889 891 893 895 897 899 901 903H1H5777P 905 907 909 911 913 915 917 919 H1H5778P 921 923 925 927 929931 933 935 H1H5780P 937 939 941 943 945 947 949 951 H1H5781P 953 955957 959 961 963 965 967 H1H5782P 969 971 973 975 977 979 981 983H1H5785B 985 987 989 991 993 995 997 999 H1H5786B 1.001 1.003 1.0051.007 1.009 1.011 1.013 1.015 H1H5788P 1.017 1.019 1.021 1.023 1.0251.027 1.029 1.031 H1H5790B 1.033 1.035 1.037 1.039 1.041 1.043 1.0451.047 H1H5791B 1.049 1.051 1.053 1.055 1.057 1.059 1.061 1.063 H1H5792B1.065 1.067 1.069 1.071 1.073 1.075 1.077 1.079 H1H5793B 1.081 1.0831.085 1.087 1.089 1.091 1.093 1.095 H1H5795B 1.097 1.099 1.101 1.1031.105 1.107 1.109 1.111 H1H5796B 1.113 1.115 1.117 1.119 1.121 1.1231.125 1.127 H1H5797B 1.129 1.131 1.133 1.135 1.137 1.139 1.141 1.143H1H5798B 1.145 1.147 1.149 1.151 1.153 1.155 1.157 1.159 H1H5799P 1.1611.163 1.165 1.167 1.169 1.171 1.173 1.175 H1H5801B 1.177 1.179 1.1811.183 1.185 1.187 1.189 1.191 H1H7194B 1.193 1.195 1.197 1.199 1.3851.387 1.389 1.391 H1H7195B 1.201 1.203 1.205 1.207 1.385 1.387 1.3891.391 H1H7196B 1.209 1.211 1.213 1.215 1.385 1.387 1.389 1.391 H1H7198B1.217 1.219 1.221 1.223 1.385 1.387 1.389 1.391 H1H7203B 1.225 1.2271.229 1.231 1.385 1.387 1.389 1.391 H1H7204B 1.233 1.235 1.237 1.2391.385 1.387 1.389 1.391 H1H7208B 1.241 1.243 1.245 1.247 1.385 1.3871.389 1.391 H1H7211B 1.249 1.251 1.253 1.255 1.385 1.387 1.389 1.391H1H7221B 1.257 1.259 1.261 1.263 1.385 1.387 1.389 1.391 H1H7223B 1.2651.267 1.269 1.271 1.385 1.387 1.389 1.391 H1H7226B 1.273 1.275 1.2771.279 1.385 1.387 1.389 1.391 H1H7232B 1.281 1.283 1.285 1.287 1.3851.387 1.389 1.391 H1H7233B 1.289 1.291 1.293 1.295 1.385 1.387 1.3891.391 H1H7241B 1.297 1.299 1.301 1.303 1.385 1.387 1.389 1.391 H1H7242B1.305 1.307 1.309 1.311 1.385 1.387 1.389 1.391 H1H7250B 1.313 1.3151.317 1.319 1.385 1.387 1.389 1.391 H1H7251B 1.321 1.323 1.325 1.3271.385 1.387 1.389 1.391 H1H7254B 1.329 1.331 1.333 1.335 1.385 1.3871.389 1.391 H1H7258B 1.337 1.339 1.341 1.343 1.385 1.387 1.389 1.391H1H7269B 1.345 1.347 1.349 1.351 1.385 1.387 1.389 1.391 H1H7279B 1.3531.355 1.357 1.359 1.385 1.387 1.389 1.391 H1xH7221G 1.361 1.363 1.3651.367 1.385 1.387 1.389 1.391 H1xH7221G3 1.369 1.371 1.373 1.375 1.3851.387 1.389 1.391 H1xH7221G5 1.377 1.379 1.381 1.383 1.385 1.387 1.3891.391

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “HIM,” “H2M,” etc.), followed by anumerical identifier (e.g. “2712,” “2692,” etc., as shown in Table 1),followed by a “P,” “N,” or “B” suffix. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1H2712N,” “H1M2692N,” “H2M2689N,” etc. The H1H, H1M and H2M prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H1H” antibody has ahuman IgG1 Fc, an “HIM” antibody has a mouse IgG1 Fc, and an “H2M”antibody has a mouse IgG2 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 1—will remain the same, and the bindingproperties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Tables 14 and 15 set out the amino acid sequence identifiers for heavychain variable regions (Table 14) and light chain variable regions(Table 15), and their corresponding CDRs, of additional anti-CD3 HCVRsand LCVRs useful in anti-PSMA×anti-CD3 bispecific antibodies of theinvention.

TABLE 14 (Heavy Chain Variable Region Amino Acid Sequences) SEQ ID NOsHeavy Chain Identifier HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1394 1396 13981400 CD3-VH-B 1410 1412 1414 1416 CD3-VH-C 1426 1428 1430 1432 CD3-VH-D1442 1444 1446 1448 CD3-VH-E 1458 1460 1462 1464 CD3-VH-F^(#) 1473 14741475 1476

TABLE 15 (Light Chain Variable Region Amino Acid Sequences) SEQ ID NOsLight Chain Identifier LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1402 1404 14061408 CD3-VL-B 1418 1420 1422 1424 CD3-VL-C 1434 1436 1438 1440 CD3-VL-D1450 1452 1454 1456 CD3-VL-E 1466 1468 1470 1472 CD3-VL-F^(#) 1477 14781479 1480

The heavy and light chain variable regions of CD3-VH-F and CD3-VL-F werederived from the anti-CD3 antibody designated “L2K” as set forth inWO2004/106380.

In addition, Tables 16 and 17 set out the sequence identifiers for thenucleotide sequences encoding the heavy chain variable regions (Table16) and light chain variable regions (Table 17), and their correspondingCDRs, of additional anti-CD3 HCVRs and LCVRs useful inanti-PSMA×anti-CD3 bispecific antibodies of the invention.

TABLE 16 (Nucleotide Sequences Encoding Heavy Chain Variable RegionSequences) SEQ ID NOs Heavy Chain Identifier HCVR HCDR1 HCDR2 HCDR3CD3-VH-AA 1393 1395 1397 1399 CD3-VH-B 1409 1411 1413 1415 CD3-VH-C 14251427 1429 1431 CD3-VH-D 1441 1443 1445 1447 CD3-VH-E 1457 1459 1461 1463

TABLE 17 (Nucleotide Sequences Encoding Light Chain Variable RegionSequences) SEQ ID NOs Light Chain Identifier LCVR LCDR1 LCDR2 LCDR3CD3-VL-AA 1401 1403 1405 1407 CD3-VL-B 1417 1419 1421 1423 CD3-VL-C 14331435 1437 1439 CD3-VL-D 1449 1451 1453 1455 CD3-VL-E 1465 1467 1469 1471

Control Constructs Used in the Following Examples

Various control constructs (anti-CD3 antibodies) were included in thefollowing experiments for comparative purposes: “OKT-3,” a mousemonoclonal antibody against human T-cell surface antigens available fromthe American Type Culture Collection (ATCC) under catalog no. CRL-8001;and “SP34,” a commercially available mouse monoclonal antibody obtained,e.g., from Biolegend, San Diego, Calif. (Cat. No. 302914) or BDPharmagen, Cat. 55052, reactive against the epsilon chain of the T3complex on human T lymphocyte cells.

Example 11: Generation of Additional Anti-CD3 Antibodies

The following procedures were aimed at identifying antibodies thatspecifically recognized CD3 (T cell co-receptor) as an antigen.

A pool of anti-CD3 antibodies were derived from a genetically modifiedmouse. Briefly, mice were immunized with a CD3 antigen and generated Bcells that comprised a diversity of human VH rearrangements in order toexpress a diverse repertoire of high-affinity antigen-specificantibodies. Antibodies described in Tables 18-21 have the same lightchain sequence of VK1-39JK5 (LCVR set forth in SEQ ID NO: 1386).

Generated antibodies were tested for affinity to human and cynomolgusmonkey CD3 antigen in an in vitro binding assay, and e.g. one CD3antibody: designated CD3-VH-P (HCVR set forth in SEQ ID NO: 1634) wasidentified, amongst a few others, that were found to bind to both humanand cynomolgus CD3 having an EC₅₀ between 1 and 40 nM affinity, asdetermined in a FACS titration of Jurkat cells and cynomolgus T cells,respectively. See, e.g. FACS binding experiments outlined in Example 5.

The germline amino acid residues of CD3-VH-P were subsequentlyidentified and an antibody designated “CD3-VH-G” was engineered tocontain only germline frameworks. Other antibody derivatives wereengineered by well-known molecular cloning techniques to replace aminoacid residues in a stepwise manner based on differences between thegermline sequence and the CD3-VH-P sequence. Each antibody derivative isgiven a “CD3-VH-G” number designation. See Table 18.

While CD3-VH-G and some other engineered antibodies retained theirbinding affinity as seen in the FACS assays, several anti-CD3 antibodiesbound to human or cyno CD3 in vitro with weak to no measurable bindingaffinity, such as 40 nM EC50. Binding affinities, binding kinetics, andother biological properties to elucidate toxicity and pharmacokinetic(pK) profiles were subsequently investigated for bispecific antibodiescomprising the exemplary anti-CD3 antibodies generated in accordancewith the methods of this Example, are described in detail in theExamples herein.

Example 12: Heavy and Light Chain Variable Regions (Amino Acid andNucleic Acid Sequences of the CDRs)

Table 18 sets forth the amino acid sequence identifiers of the heavychain variable regions and CDRs of selected anti-CD3 antibodies of theinvention. The corresponding nucleic acid sequence identifiers are setforth in Table 19.

Amino acid and nucleic acid sequences were determined for each antibodyheavy chain sequence. Each antibody heavy chain derived from thegermline sequence (SEQ ID NO: 1662) was assigned a “G” numberdesignation for consistent nomenclature. Table 2 sets forth the aminoacid sequence identifiers of the heavy chain variable regions and CDRsof engineered anti-CD3 antibodies of the invention. The correspondingnucleic acid sequence identifiers are set forth in Table 19. The aminoacid and nucleic acid sequence identifiers of the light chain variableregion and CDRs are also identified below in Tables 20 and 21,respectively.

TABLE 18 Heavy Chain Amino Acid Sequence Identifiers Antibody CD3-VH SEQID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G  1482 1484 1486 1488CD3-VH-G2  1490 1492 1494 1496 CD3-VH-G3  1498 1500 1502 1504 CD3-VH-G4 1506 1508 1510 1512 CD3-VH-G5  1514 1516 1518 1520 CD3-VH-G8  1522 15241526 1528 CD3-VH-G9  1530 1532 1534 1536 CD3-VH-G10 1538 1540 1542 1544CD3-VH-G11 1546 1548 1550 1552 CD3-VH-G12 1554 1556 1558 1560 CD3-VH-G131562 1564 1566 1568 CD3-VH-G14 1570 1572 1574 1576 CD3-VH-G15 1578 15801582 1584 CD3-VH-G16 1586 1588 1590 1592 CD3-VH-G17 1594 1596 1598 1600CD3-VH-G18 1602 1604 1606 1608 CD3-VH-G19 1610 1612 1614 1616 CD3-VH-G201618 1620 1622 1624 CD3-VH-G21 1626 1628 1630 1632 CD3-VH-P   1634 16361638 1640

TABLE 19 Heavy Chain Nucleic Acid Sequence Identifiers Antibody CD3-VHSEQ ID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G  1481 1483 14851487 CD3-VH-G2  1489 1491 1493 1495 CD3-VH-G3  1497 1499 1501 1503CD3-VH-G4  1505 1507 1509 1511 CD3-VH-G5  1513 1515 1517 1519 CD3-VH-G8 1521 1523 1525 1527 CD3-VH-G9  1529 1531 1533 1535 CD3-VH-G10 1537 15391541 1543 CD3-VH-G11 1545 1547 1549 1551 CD3-VH-G12 1553 1555 1557 1559CD3-VH-G13 1561 1563 1565 1567 CD3-VH-G14 1569 1571 1573 1575 CD3-VH-G151577 1579 1581 1583 CD3-VH-G16 1585 1587 1589 1591 CD3-VH-G17 1593 15951597 1599 CD3-VH-G18 1601 1603 1605 1607 CD3-VH-G19 1609 1611 1613 1615CD3-VH-G20 1617 1619 1621 1623 CD3-VH-G21 1625 1627 1629 1631 CD3-VH-P  1633 1635 1637 1639

TABLE 20 Light Chain Amino Acid Sequence Identifiers Antibody SEQ IDNOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 1386 1388 1390 1392

TABLE 21 Light Chain Nucleic Acid Sequence Identifiers Antibody SEQ IDNOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 1385 1387 1389 1391

Control 1 antibody designated “CD3-L2K” was constructed based on a knownanti-CD3 antibody (i.e., the anti-CD3 antibody “L2K” as set forth inWO2004/106380).

Isotype Control Antibody, referred to in the Examples herein, is anisotype matched (modified IgG4) antibody that interacts with anirrelevant antigen, i.e. FelD1 antigen.

Example 13: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-CD3 Antibodies

Binding affinities and kinetic constants of human monoclonal anti-CD3antibodies were determined by surface plasmon resonance at 25° C. usingeither an antibody-capture format (Tables 22, 24, and 26) or anantigen-capture format (Tables 23, 25, and 27). Measurements wereconducted on a T200 Biacore™ instrument.

In the antibody-capture format, the Biacore™ sensor surface wasderivatized with a rabbit anti-mouse Fc for hybridoma capture (antibodyprefix H1M or H2M) or a mouse anti-human Fc surface for human IgGformatted antibodies (antibody prefix H1H). Soluble heterodimeric CD3protein (hCD3-epsilon/hCD3-delta; SEQ ID NOs:1652/1653) with either ahuman Fc tag (hFcΔAdp/hFc; SEQ ID NOs:1683/1684) or a mouse Fc tag(mFcΔAdp/mFc; SEQ ID NOs:1685/1686) was injected over the antibodycaptured surface and the binding response was recorded. HeterodimericCD3 protein was purified using the method described in Davis et al.(US2010/0331527).

In the antigen-capture format, heterodimeric CD3 protein was capturedusing a rabbit anti-mouse Fc or mouse anti-human Fc and the respectiveantibodies were injected over the captured antigen.

Antibodies were analyzed in their conventional divalent format (Tables22-25) or in a monovalent 1-arm configuration (Tables 26-27) in whichthe second Fab from the antibody was removed and only the Fc portion(CH2-CH3) was expressed.

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t_(1/2) (min)=(In2/(60*k_(d)). NT=not tested; NB=no bindingobserved.

TABLE 22 Biacore ™ Binding Affinities of Hybridoma mAbs (H1M and H2M)Binding at 25° C./Antibody-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) T½ (min) H2M2689N 7.73E+05 3.23E−03 4.18E−09 4 H2M2690N9.70E+03 2.02E−04 2.09E−08 57 H2M2691N 1.03E+04 2.07E−04 2.01E−08 56H1M2692N 8.05E+03 4.34E−04 5.39E−08 27 H2M2704N 3.46E+04 6.92E−042.00E−08 17 H2M2705N 6.62E+04 9.10E−04 1.37E−08 13 H2M2706N 3.29E+044.44E−03 1.35E−07 3 H2M2707N 2.95E+04 1.87E−03 6.35E−08 6 H2M2708N6.94E+04 6.12E−04 8.82E−09 19 H2M2709N NT NT NT NT H2M2710N 6.72E+047.53E−04 1.12E−08 15 H2M2711N 6.72E+04 7.67E−04 1.14E−08 15 H1M2712N9.32E+03 2.19E−04 2.35E−08 53 H2M2774N 7.79E+04 9.18E−04 1.18E−08 13H2M2775N 6.97E+04 6.26E−04 8.98E−09 18 H2M2776N 6.29E+04 6.39E−041.02E−08 18 H2M2777N 3.70E+04 1.63E−03 4.39E−08 7 H2M2778N 2.13E+041.89E−04 8.90E−09 61 H2M2779N 2.18E+04 2.28E−04 1.05E−08 51 H2M2789N NTNT NT NT H2M2862N 3.72E+04 3.00E−03 8.07E−08 4 H2M2885N 6.82E+046.51E−04 9.54E−09 18 H2M2886N 7.29E+04 6.53E−04 8.96E−09 18 H2M3540N3.77E+04 6.11E−04 1.62E−08 19 H2M3541N 7.10E+03 1.35E−03 1.89E−07 9H1M3542N 2.37E+04 5.08E−04 2.14E−08 23 H2M3543N 7.53E+03 2.26E−043.00E−08 51 H1M3544N 9.69E+03 1.42E−04 1.46E−08 82 H2M3547N 2.18E+043.47E−04 1.59E−08 33 H2M3548N 3.87E+04 5.04E−03 1.30E−07 2 H1M3549N1.18E+04 9.19E−04 7.76E−08 13 H2M3563N 3.24E+04 1.19E−04 3.66E−09 97H1M3613N 1.93E+04 3.04E−04 1.57E−08 38

TABLE 23 Biacore ™ Binding Affinities of Hybridoma mAbs (H1M and H2M)Binding at 25° C./Antigen-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) T½ (min) H2M2689N 1.71E+06 9.97E−05 5.83E−11 116 H2M2690N7.51E+04 6.35E−06 7.99E−11 1820 H2M2691N 3.94E+04 9.98E−06 2.54E−10 1158H1M2692N 4.19E+04 9.90E−06 2.38E−10 1167 H2M2704N 1.32E+06 2.48E−041.87E−10 47 H2M2705N 2.43E+06 3.41E−04 1.40E−10 34 H2M2706N 5.63E+053.06E−04 5.44E−10 38 H2M2707N 3.99E+05 2.85E−04 7.15E−10 41 H2M2708N1.73E+06 2.27E−04 1.31E−10 51 H2M2709N NT NT NT NT H2M2710N 1.59E+062.43E−04 1.53E−10 48 H2M2711N 1.59E+06 2.40E−04 1.51E−10 48 H1M2712N4.75E+04 1.37E−05 2.95E−10 846 H2M2774N 2.49E+06 3.36E−04 1.35E−10 34H2M2775N 1.56E+06 2.16E−04 1.38E−10 53 H2M2776N 1.58E+06 2.22E−041.40E−10 52 H2M2777N 5.80E+05 3.21E−04 5.54E−10 36 H2M2778N 1.50E+056.57E−06 4.68E−11 1758 H2M2779N 1.28E+05 1.23E−05 9.38E−11 941 H2M2789NNT NT NT NT H2M2862N 5.91E+05 3.21E−04 5.41E−10 36 H2M2885N 1.37E+061.52E−04 1.11E−10 76 H2M2886N 1.42E+06 1.36E−04 9.56E−11 85 H2M3540N2.55E+06 5.87E−04 2.31E−10 20 H2M3541N 8.40E+04 1.16E−03 1.38E−08 10H1M3542N 4.37E+05 2.00E−04 4.57E−10 58 H2M3543N 1.22E+05 7.96E−056.53E−10 145 H1M3544N 5.74E+04 5.98E−05 1.04E−09 193 H2M3547N 4.70E−051.00E−05 2.15E−11 1155 H2M3548N NT NT NT NT H1M3549N 2.81E+05 2.89E−041.03E−09 40 H2M3563N 6.16E+05 4.77E−05 7.73E−11 242 H1M3613N 2.20E+059.60E−05 4.35E−10 120

TABLE 24 Biacore ™ Binding Affinities of Human Fc mAbs (H1H) Binding at25° C./Antibody-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar)T½ (min) H1H2690N NT NT NT NT H1H2712N 3.06E+03 2.70E−04 8.82E−08 43H1H5751P 4.01E+03 5.18E−04 1.29E−07 22 H1H5752P NB NB NB NB H1H5753B NTNT NT NT H1H5755B 8.21E+03 4.72E−04 5.75E−08 24 H1H5756B 8.15E+032.66E−04 3.26E−08 43 H1H5757B 6.63E+03 7.85E−04 1.18E−07 15 H1H5758B5.02E+03 1.17E−03 2.33E−07 10 H1H5761P 4.72E+03 2.44E−02 5.16E−06 0H1H5763P 1.85E+04 5.40E−02 2.92E−06 0 H1H5764P 4.16E+03 1.59E−023.82E−06 1 H1H5769P 7.80E+03 9.41E−04 1.21E−07 12 H1H5771P 3.00E+046.26E−04 2.09E−08 18 H1H5772S 1.56E+04 1.55E−03 9.96E−08 7 H1H5777P1.35E+04 3.02E−03 2.24E−07 4 H1H5778P 5.52E+03 1.54E−04 2.78E−08 75H1H5780P 1.31E+04 3.99E−04 3.04E−08 29 H1H5781P 8.61E+03 4.97E−045.77E−08 23 H1H5782P NB NB NB NB H1H5785B NT NT NT NT H1H5786B 1.26E+041.08E−03 8.54E−08 11 H1H5788P 2.88E+03 2.91E−04 1.01E−07 40 H1H5790B1.82E+04 5.17E−04 2.83E−08 22 H1H5791B 1.09E+04 7.90E−04 7.25E−08 15H1H5792B NT NT NT NT H1H5793B 8.54E+03 3.82E−04 4.47E−08 30 H1H5795B1.73E+04 5.76E−04 3.33E−08 20 H1H5796B 1.47E+04 8.91E−04 6.05E−08 13H1H5797B NT NT NT NT H1H5798B NT NT NT NT H1H5799P 1.36E+04 7.88E−035.79E−07 1 H1H5801B 6.57E+03 1.62E−03 2.46E−07 7 OKT3 2.10E+06 2.00E+001.00E−06 0.35 sec

TABLE 25 Biacore ™ Binding Affinities of Human Fc mAbs (H1H) Binding at25° C./Antigen-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar)T½ (min) H1H2690N NT NT NT NT H1H2712N 8.93E+04 8.68E−05 9.71E−10 133H1H5751P 7.24E+04 2.47E−04 3.42E−09 47 H1H5752P NB NB NB NB H1H5753B NTNT NT NT H1H5755B 2.15E+05 2.01E−04 9.36E−10 57 H1H5756B 1.44E+051.11E−04 7.67E−10 105 H1H5757B 1.80E+05 2.95E−04 1.64E−09 39 H1H5758B1.42E+05 5.62E−04 3.97E−09 21 H1H5761P 2.11E+05 1.13E−02 5.34E−08 1H1H5763P 1.84E+05 1.70E−02 9.24E−08 1 H1H5764P 3.50E+05 7.36E−032.10E−08 2 H1H5769P 1.19E+05 5.23E−04 4.41E−09 22 H1H5771P 9.23E+053.42E−04 3.71E−10 34 H1H5772S 5.19E+05 8.69E−04 1.67E−09 13 H1H5777P4.83E+05 1.70E−03 3.52E−09 7 H1H5778P 3.99E+05 3.42E−05 8.56E−11 338H1H5780P 4.78E+05 1.71E−04 3.58E−10 68 H1H5781P 1.40E+05 2.68E−041.92E−09 43 H1H5782P NB NB NB NB H1H5785B NT NT NT NT H1H5786B 3.00E+064.24E−04 1.41E−10 27 H1H5788P 7.06E+04 1.64E−04 2.33E−09 70 H1H5790B9.25E+05 2.36E−04 2.54E−10 49 H1H5791B 7.86E+05 3.40E−04 4.33E−10 34H1H5792B NT NT NT NT H1H5793B 4.78E+05 1.59E−04 3.33E−10 73 H1H5795B1.58E+06 2.29E−04 1.45E−10 50 H1H5796B 1.05E+05 2.44E−04 2.32E−09 47H1H5797B NT NT NT NT H1H5798B NT NT NT NT H1H5799P 7.18E+05 5.64E−037.85E−09 2 H1H5801B 3.31E+05 1.12E−03 3.38E−09 10 OKT3 3.94E+06 2.18E−025.53E−09 0.5

TABLE 26 Biacore ™ Binding Affinities of monovalent 1-arm mAbs Bindingat 25° C./Antibody-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) T½ (min) H1H7194P 1.16E+04 1.51E−04 1.30E−08 76 H1H7195P3.13E+04 9.89E−05 3.16E−09 117 H1H7196P 1.07E+04 4.43E−04 4.13E−08 26H1H7198P 2.63E+04 1.58E−04 6.02E−09 73 H1H7203P 1.46E+04 2.67E−041.83E−08 43 H1H7204P 1.43E+04 3.62E−04 2.53E−08 32 H1H7208P NT NT NT NTH1H7211P 1.41E+04 1.59E−04 1.13E−08 73 H1H7221P 1.07E+04 2.92E−042.75E−08 40 H1H7223P 1.60E+04 3.07E−04 1.92E−08 38 H1H7226P 1.30E+043.55E−04 2.72E−08 33 H1H7232P 8.03E+03 1.77E−03 2.20E−07 7 H1H7233P1.11E+04 2.69E−04 2.42E−08 43 H1H7241P 1.34E+04 2.95E−04 2.20E−08 39H1H7242P 2.15E+04 6.64E−04 3.09E−08 17 H1H7250P 2.34E+04 2.47E−041.05E−08 47 H1H7251P 2.56E+04 1.07E−03 4.17E−08 11 H1H7254P 2.60E+043.88E−04 1.49E−08 30 H1H7258P 1.26E+04 3.02E−04 2.40E−08 38 H1H7269P2.57E+04 6.24E−03 2.43E−07 2 H1H7279P NB NB NB NB H1xH7221G NT NT NT NTH1xH7221G3 NB NB NB NB H1xH7221G5 NB NB NB NB

TABLE 27 Biacore ™ Binding Affinities of monovalent 1-arm mAbs Bindingat 25° C./Antigen-Capture Format Antibody ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) T½ (min) H1H7194P 3.50E+05 8.43E−05 2.41E−10 137 H1H7195P5.66E+05 7.14E−05 1.26E−10 162 H1H7196P 1.85E+05 4.61E−04 2.49E−09 25H1H7198P 6.28E+05 7.07E−05 1.12E−10 163 H1H7203P 4.79E+05 2.38E−044.98E−10 48 H1H7204P 1.73E+05 3.65E−04 2.12E−09 32 H1H7208P NT NT NT NTH1H7211P 3.45E+05 9.61E−05 2.79E−10 120 H1H7221P 1.36E+05 2.39E−041.75E−09 48 H1H7223P 1.87E+05 2.86E−04 1.53E−09 40 H1H7226P 4.18E+052.36E−04 5.65E−10 49 H1H7232P 1.49E+05 1.49E−03 1.00E−08 8 H1H7233P1.61E+05 2.04E−04 1.27E−09 57 H1H7241P 1.87E+05 2.36E−04 1.26E−09 49H1H7242P 3.83E+05 1.01E−03 2.63E−09 11 H1H7250P 2.31E+05 1.89E−048.20E−10 61 H1H7251P 4.47E+05 1.19E−03 2.67E−09 10 H1H7254P 4.33E+053.30E−04 7.62E−10 35 H1H7258P 1.33E+05 2.90E−04 2.18E−09 40 H1H7269P2.77E+05 6.89E−03 2.49E−08 2 H1H7279P NB NB NB NB H1xH7221G NT NT NT NTH1xH7221G3 NB NB NB NB H1xH7221G5 NB NB NB NB

As shown in Tables 22-27, Several anti-CD3 antibodies of the presentinvention bind CD3, in either antibody-capture or antigen-captureformats, with high affinity.

Example 14: Anti-CD3 Antibodies Bind and Proliferate Human T-Cells

Anti-CD3 antibodies of the present invention were tested for theirability to bind to human T-cells and induce their proliferation. Bindingwas assessed using Jurkat cells (a CD3+ human T-cell line), whileproliferation of Peripheral Blood Mononuclear Cells (PBMC) was assessedusing ATP catalyzed quantification (CellTiter Glo®). Anti-CD3 antibodyOKT3 acted as a positive control and irrelevant isotype matchedantibodies served as negative controls.

FACS data was acquired using the following protocol: Cells at 2×10⁵ perwell were incubated with serially diluted antibodies for 30 min on ice.Post incubation, cells were washed and secondary antibody was added andincubated for an additional 30 minutes. After incubation, cells werewashed, re-suspended in cold PBS containing 1% BSA and analyzed by flowcytometry with viable Jurkat cells gated by side and forward scatters.The EC₅₀s for cell binding titration were determined using Prismsoftware with values calculated using a 4-parameter non-linearregression analysis.

Proliferation data was acquired using the following protocol: Human PBMC(5×10⁴/well) were incubated with a 3-fold serial dilution of anti-CD3and a fixed concentration of a commercial anti-CD28 antibody (200 ng/ml)in 96 well plates for 72 h at 37° C. Following incubation, CellTiterGlo® was added and luminescence was measured using a VICTOR X5multi-label plate reader (PerkinElmer). The EC₅₀ of cell viability (ATPcatalyzed quantification) was calculated using a 4-parameter non-linearregression analysis in GraphPad Prism.

Results of the binding and proliferation experiments are summarized inTables 28-30.

TABLE 28 Hybridoma Anti-CD3 mAbs Bind & Proliferate Human T-Cells EC₅₀[M] EC₅₀ [M] hPBMC Antibody FACS JURKAT Proliferation H2M2689N NB0.00E+00 H2M2690N 4.37E−09 5.37E−12 H2M2691N 6.77E−09 3.43E−11 H1M2692N5.99E−09 1.42E−10 H2M2704N 8.45E−10 2.93E−12 H2M2705N 2.96E−10 1.76E−11H2M2706N 2.37E−09 3.86E−12 H2M2707N 1.24E−07 1.92E−12 H2M2708N 6.58E−102.69E−08 H2M2709N 7.11E−10 2.48E−11 H2M2710N 7.10E−10 2.11E−10 H2M2711N1.16E−09 6.48E−10 H1M2712N 2.19E−08 1.28E−10 H2M2774N 3.52E−10 4.92E−10H2M2775N 1.32E−09 1.09E−09 H2M2776N 4.91E−10 2.84E−11 H2M2777N 2.16E−092.51E−11 H2M2778N 3.62E−09 0.00E+00 H2M2779N NT 0.00E+00 H2M2789N NT2.85E−08 H2M2862N 7.68E−09 6.72E−13 H2M2885N 2.09E−09 2.49E−12 H2M2886N3.97E−09 2.69E−12 H2M3540N 3.99E−09 3.16E−12 H2M3541N 3.70E−09 6.40E−12H1M3542N 2.01E−09 0.00E+00 H2M3543N 5.63E−09 6.12E−12 H1M3544N 2.32E−080.00E+00 H2M3547N 2.71E−09 5.02E−12 H2M3548N 1.10E−09 1.89E−12 H1M3549N2.30E−09 0.00E+00 H2M3563N 1.07E−09 7.74E−12 H1M3613N 1.03E−08 0.00E+00Isotype Ctrl NB 0.00E+00 NB: No Binding; NT: Not Tested

TABLE 29 Human Fc Anti-CD3 mAbs Bind & Proliferate Human T-Cells EC₅₀[M] EC₅₀ [M] hPBMC Antibody FACS JURKAT Proliferation H1H5751P 2.12E−099.29E−12 H1H5752P 3.43E−10 1.09E−12 H1H5753B NB 9.14E−11 H1H5755B1.23E−09 4.24E−12 H1H5756B NB 0.00E+00 H1H5757B 3.38E−09 4.86E−12H1H5758B 1.90E−09 2.13E−12 H1H5761P 2.10E−09 3.62E−13 H1H5763P 2.76E−093.11E−13 H1H5764P 8.80E−10 3.27E−13 H1H5769P 4.10E−09 6.17E−12 H1H5771PNT 6.35E−12 H1H5772S 6.64E−10 4.42E−12 H1H5777P 5.71E−10 3.04E−12H1H5778P 6.85E−10 5.04E−12 H1H5780P 7.62E−10 3.44E−12 H1H5781P 1.23E−096.08E−12 H1H5782P NB 5.17E−12 H1H5785B NB 0.00E+00 H1H5786B 1.10E−091.79E−12 H1H5788P 3.53E−09 4.62E−12 H1H5790B 3.55E−09 2.71E−12 H1H5791B3.77E−09 1.75E−12 H1H5792B 5.87E−09 6.47E−12 H1H5793B 4.62E−09 3.28E−12H1H5795B 2.04E−09 3.09E−12 H1H5796B 9.82E−09 4.37E−12 H1H5797B 3.96E−081.07E−11 H1H5798B 5.57E−09 2.59E−12 H1H5799P NT 1.63E−13 H1H5801B1.55E−08 1.09E−12 OKT3 1.96E−10 3.30E−13 Isotype Ctrl NB 0.00E+00 NB: NoBinding; NT: Not Tested

TABLE 30 Monovalent 1-arm Anti-CD3 mAbs Bind & Proliferate Human T-CellsEC₅₀ [M] EC₅₀ [M] hPBMC Antibody FACS JURKAT Proliferation H1H7194P1.50E−09 2.37E−12 H1H7195P 3.42E−10 2.42E−12 H1H7196P 3.44E−08 1.27E−12H1H7198P 7.26E−10 2.55E−12 H1H7203P 3.24E−09 1.64E−12 H1H7204P 2.29E−091.51E−12 H1H7208P 5.19E−08 1.46E−12 H1H7211P 7.01E−10 2.75E−12 H1H7221P1.40E−09 2.60E−12 H1H7223P 9.37E−10 1.07E−12 H1H7226P 7.95E−10 9.52E−13H1H7232P 1.50E−09 1.03E−12 H1H7233P 7.15E−10 7.34E−13 H1H7241P 1.01E−091.05E−12 H1H7242P 1.83E−09 2.13E−12 H1H7250P 1.37E−09 2.43E−12 H1H7251P1.45E−09 1.30E−12 H1H7254P 1.09E−09 2.80E−12 H1H7258P 1.07E−09 2.17E−12H1H7269P 1.95E−09 1.15E−12 H1H7279P NB 0.00E+00 Isotype Ctrl NB 0.00E+00NB: No Binding; NT: Not Tested

As shown in Tables 7-9, the vast majority of anti-CD3 antibodies of theinvention bound human T-cells and induced T-cell proliferation.

Example 15: Anti-CD3 Antibodies Bind and Proliferate Monkey T-Cells

A subset of anti-CD3 antibodies of the invention was tested for theability to bind to and induce proliferation of monkey T-cells.

FACS data was acquired using the following protocol: Cells at 2×10⁵ perwell were incubated with serially diluted antibodies for 30 min on ice.Post incubation, cells were washed and secondary antibodies were addedand incubated for an additional 30 minutes. After incubation, cells werewashed, re-suspended in cold PBS containing 1% BSA and analyzed by flowcytometry. CD4+ monkey T cells were gated by side and forward scatters,and on the CD2+CD4+CD20-population. The EC₅₀s for cell binding titrationwere calculated using a 4-parameter non-linear regression analysis inGraphPad Prism.

Proliferation data was acquired using the following protocol: Freshlyisolated cynomolgus monkey derived PBMC (5×10⁴/well) were incubated witha 3-fold serial dilution of anti-CD3 antibody and a fixed concentrationof a commercial anti-CD28 antibody (500 ng/ml) antibody in 96 wellplates for 72 h at 37° C. Following incubation, CellTiter Glo® was addedand luminescence was measured using a VICTOR X5 multi-label plate reader(PerkinElmer). The EC₅₀ of cell viability (ATP catalyzed quantification)was calculated using a 4-parameter non-linear regression analysis inGraphPad Prism.

Results of the binding and proliferation experiments are summarized inTables 31 and 32.

TABLE 31 Anti-CD3 mAbs Bind & Proliferate monkey PBMCs EC50 [M] EC50 [M]mfPBMC Antibody FACS PBMCs Proliferation H1H2690N 5.66E−09 2.71E−12H1H2712N 2.29E−09 2.72E−12 H2M3547N 1.12E−10 NT H2M3563N 1.65E−10 NTH1H5761P NT 2.81E−09 H1H5763P NT 0.00E+00 H1H5764P NT 4.06E−10 H1H5769PNT 8.33E−13 H1H5771P NT 2.74E−12 H1H5772S NT 1.47E−12 H1H5778P NT5.93E−13 H1H5780P NT 3.13E−13 H1H5781P NT 7.92E−13 H1H5788P NT 2.01E−12OKT3 NB NT SP34 7.03E−11 1.71E−12 NB: No Binding; NT: not tested

TABLE 32 Monovalent 1-arm Anti-CD3 mAbs Bind & Proliferate Monkey PBMCsEC50 [M] EC50 [M] mfPBMC Antibody FACS PBMCs Proliferation H1H7194P NT4.84E−12 H1H7195P NT 1.36E−12 H1H7196P NT 1.40E−08 H1H7198P NT 2.29E−12H1H7203P NT 4.97E−13 H1H7204P NT 1.26E−11 H1H7208P NT 7.02E−12 H1H7211PNT 2.81E−13 H1H7221P NT 1.72E−12 H1H7223P NT 6.75E−11 H1H7226P NT2.26E−11 H1H7232P NT 4.90E−11 H1H7233P NT 4.35E−12 H1H7241P NT 2.05E−11H1H7242P NT 1.38E−11 H1H7250P NT 7.27E−11 H1H7251P NT 1.83E−11 H1H7254PNT 8.88E−11 H1H7258P NT 1.11E−11 NB: No Binding; NT: not tested

As shown in Tables 31 and 32, several anti-CD3 antibodies of theinvention bound CD2+CD4+ monkey T-cells and induced their proliferation.OKT3 did not drive monkey PBMC proliferation, while SP34 was activeagainst monkey PBMCs.

Example 16: Anti-CD3 mAbs Support T-Cell-Mediated Killing of Tumor Cells

The ability of anti-CD3 antibodies to redirect T-cell mediated killingvia Fc/FcR interactions was studied using a calcein based U937 killingassay. Briefly, human PBMC were isolated over Ficoll-Paque and activatedover a course of several days with media containing human IL-2 (30 U/ml)and T-cell activation beads (anti-CD3/CD28). U937 cells were labeledwith calcein, and then incubated with activated T-cells at a 10:1effector: target ratio using 3-fold serial dilutions of antibodies overa course of 3 hours at 37° C. Following incubation, the plates werecentrifuged and supernatants were transferred to a translucent blackclear bottom plate for fluorescence analysis. EO₅₀ values, defined asthe molar concentration of CD3 antibody that induces 50% cytotoxicity,were calculated using a 4-parameter non-linear regression analysis inGraphPad Prism. Results using hybridoma antibodies, human Fc antibodies,and monovalent one-arm antibodies are shown in Tables 33, 34 and 35,respectively.

TABLE 33 Hybridoma Anti-CD3 mAbs Redirect T-Cell Killing to U937 CellsU937 Cytotoxicity Antibody Human T-cells [M] H2M2689N 0.00E+00 H2M2690N2.79E−11 H2M2691N 2.34E−11 H1M2692N 3.59E−10 H2M2704N 2.49E−12 H2M2705N1.73E−12 H2M2706N 7.91E−12 H2M2707N 7.21E−12 H2M2708N 3.27E−12 H2M2709N3.47E−12 H2M2710N 3.97E−12 H2M2711N 3.66E−12 H1M2712N 3.14E−10 H2M2774N2.46E−12 H2M2775N 3.38E−12 H2M2776N 4.06E−12 H2M2777N 4.86E−12 H2M2778N0.00E+00 H2M2779N 6.75E−10 H2M2789N NT H2M2862N 7.66E−12 H2M2885N3.71E−12 H2M2886N 8.06E−12 H2M3540N 1.25E−11 H2M3541N 5.39E−11 H1M3542N2.92E−11 H2M3543N 1.31E−11 H1M3544N 1.72E-10 H2M3547N 3.17E-11 H2M3548N5.50E-12 H1M3549N 1.07E−10 H2M3563N 4.05E−11 H1M3613N 8.66E−10 IsotypeCtrl 0.00E+00 NT: Not Tested

TABLE 34 Human Fc formatted Anti-CD3 mAbs Redirect T-Cell Killing toU937 Cells U937 Cytotoxicity Antibody Human T-cells [M] H1H5751P1.30E−10 H1H5752P 1.85E−11 H1H5753B 3.79E−10 H1H5755B 5.16E−11 H1H5756B7.69E−11 H1H5757B 9.65E−11 H1H5758B 8.86E−08 H1H5761P 2.00E−12 H1H5763PNT H1H5764P NT H1H5769P 5.65E−11 H1H5771P NT H1H5772S 6.89E−13 H1H5777P4.87E−13 H1H5778P 3.41E−13 H1H5780P 4.03E−12 H1H5781P 1.83E−12 H1H5782P5.18E−12 H1H5785B 4.43E−11 H1H5786B 6.10E−11 H1H5788P 1.54E−11 H1H5790B8.71E−11 H1H5791B 8.01E−11 H1H5792B 1.40E−10 H1H5793B 8.85E−11 H1H5795B6.74E−11 H1H5796B 5.03E−10 H1H5797B 5.76E−10 H1H5798B 1.81E−10 H1H5799PNT H1H5801B 9.23E−11 OKT3 2.35E−12 Isotype Ctrl 0.00E+00 NT: Not Tested

TABLE 35 Monovalent 1-arm Anti-CD3 mAb Redirect T-Cell Killing to U937Cells U937 Cytotoxicity Antibody Human T-cells [M] H1H7194P 4.71E−12H1H7195P 6.10E−12 H1H7196P 1.96E−11 H1H7198P 5.21E−12 H1H7203P 5.47E−12H1H7204P 1.08E−11 H1H7208P 4.59E−11 H1H7211P 7.89E−12 H1H7221P 9.21E−12H1H7223P 5.30E−12 H1H7226P 1.04E−11 H1H7232P 9.96E−12 H1H7233P 1.19E−11H1H7241P 1.23E−11 H1H7242P 7.50E−12 H1H7250P 5.91E−12 H1H7251P 1.81E−12H1H7254P 4.18E−12 H1H7258P 1.53E−11 H1H7269P 1.08E−11 H1H7279P 0.00E+00Isotype Ctrl 0.00E+00 NT: Not Tested

As shown in Tables 33-35, most anti-CD3 antibodies, as well as OKT3,supported redirected T-cell mediated killing in this assay system. Theobserved killing, believed to be dependent on the antibody's Fcengagement with the Fc Receptor on U937 cells leading to clustering ofCD3 on adjacent T-cells, was squelched by addition of non-specific humanIgG (data not shown).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragment thereof that binds human prostate-specific membrane antigen (PSMA) with a binding dissociation equilibrium constant (K_(D)) value of less than about 80 nM as measured in a surface plasmon resonance assay at 37° C.
 2. An isolated antibody or antigen-binding fragment thereof that binds human PSMA with a dissociative half-life (t½) of greater than about 10 minutes as measured in a surface plasmon resonance assay at 37° C.
 3. The antibody or antigen-binding fragment of any one of claims 1 to 2, wherein the antibody or antigen-binding fragment thereof competes for binding to human PSMA with a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table
 1. 4. The antibody or antigen-binding fragment of claim 3, wherein the reference antibody comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.
 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human PSMA as a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table
 1. 6. The antibody or antigen-binding fragment of claim 5, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human PSMA as a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.
 7. An isolated antibody or antigen-binding fragment thereof that binds human PSMA, wherein the antibody or antigen-binding fragment comprises: (a) the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and (b) the CDRs of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table
 1. 8. The isolated antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs:2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.
 9. The isolated antibody or antigen-binding fragment of claim 8, wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NOs: 4-6-8-1644-1646-1648; 12-14-16-1644-1646-1648; 20-22-24-1644-1646-1648; 28-30-32-1644-1646-1648; 36-38-40-1644-1646-1648; 44-46-48-1644-1646-1648; 52-54-56-1644-1646-1648; 60-62-64-1644-1646-1648; 68-70-72-1644-1646-1648; 76-78-80-1644-1646-1648; 84-86-88-1644-1646-1648; 92-94-96-1644-1646-1648; 100-102-104-1644-1646-1648; 108-110-112-1644-1646-1648; 116-118-120-1644-1646-1648; 124-126-128-132-134-136; and 140-142-144-148-150-152.
 10. An isolated antibody or antigen-binding fragment thereof that binds human PSMA, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 138; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 and
 146. 11. The isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs:2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642; 114/1642; 122/130; and 138/146.
 12. A bispecific antigen-binding molecule comprising a first antigen-binding domain that binds human CD3 and a second antigen-binding domain that binds human PSMA, wherein the second antigen-binding domain is derived from the antibody or antigen-binding fragment of any one of claims 1-11.
 13. A bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds human CD3, and a second antigen-binding domain that specifically binds human PSMA.
 14. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the first antigen-binding domain binds human cells expressing human CD3 and cynomolgus monkey cells expressing cynomolgus CD3.
 15. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the second antigen-binding domain binds human cells expressing human PSMA and cynomolgus monkey cells expressing cynomolgus PSMA.
 16. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the antigen-binding molecule binds both human CD3 and human PSMA and induces T cell-mediated cell killing of PSMA-expressing cells.
 17. The bispecific antigen-binding molecule of claim 12 or claim 13 wherein the antigen-binding molecule inhibits tumor growth in immunocompromised mice bearing human prostate cancer xenografts.
 18. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the antigen-binding molecule inhibits tumor growth in immunocompetent mice bearing human prostate cancer xenografts.
 19. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the antigen-binding molecule suppresses tumor growth of established tumors in immunocompromised mice bearing human prostate cancer xenografts.
 20. The bispecific antigen-binding molecule of claim 12 or claim 13, wherein the antigen-binding molecule reduces tumor growth of established tumors in immunocompetent mice bearing human prostate cancer xenografts.
 21. The bispecific antigen-binding molecule of any one of claims 12-20, wherein the antigen-binding molecule induces T cell-mediated tumor cell killing with an EO₅₀ value of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay.
 22. A bispecific antigen-binding molecule of any one of claims 12-21, wherein the second antigen-binding domain specifically binds human PSMA with an K_(D) value of less than about 80 nM, as measured in an in vitro surface plasmon resonance binding assay.
 23. The bispecific antigen-binding molecule of any one of claims 12-22, wherein the second antigen-binding domain specifically binds each of human PSMA with an K_(D) value of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, or less than about 600 pM, as measured in an in vitro surface plasmon resonance binding assay.
 24. The bispecific antigen-binding molecule of any one of claims 12-23 that is a bispecific antibody or bispecific antigen-binding fragment thereof.
 25. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the second antigen-binding domain that specifically binds human PSMA comprises the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, and 138; and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 930 and
 1642. 26. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the second antigen-binding domain that specifically binds human PSMA comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, and 140; A2-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, and 142; A2-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, and 144; A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 932 and 1644; A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 934 and 1646; and A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 936 and
 1648. 27. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the second antigen-binding domain that specifically binds human PSMA comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 122/162, 122/930 and 66/1642.
 28. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence as set forth in Table 12, Table 14, or Table 18 and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence as set forth in Table 12, Table 15, or Table
 20. 29. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 922, 154, 1482, 1490, 1498, 1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578, 1586, 1594, 1602, 1610, 1618, 1626, and 1634, and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 930 and
 1642. 30. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 924, 156; 1484, 1492, 1500, 1508, 1516, 1524, 1532, 1540, 1548, 1556, 1564, 1572, 1580, 1588, 1596, 1604, 1612, 1620, 1628, and 1636; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 926, 158, 1486, 1494, 1502, 1510, 1518, 1526, 1534, 1542, 1550, 1558, 1566, 1574, 1582, 1590, 1598, 1606, 1614, 1622, 1630, and 1638; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 928, 160, 1488, 1496, 1504, 1512, 1520, 1528, 1536, 1544, 1552, 1560, 1568, 1576, 1584, 1592, 1600, 1608, 1616, 1624, 1632, and 1640; A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 932, 164, and 1644; A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 934 and 1646; and A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 936 and
 1648. 31. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 922/930, 154/162, 1482/1642, 1490/1642, 1498/1642, 1506/1642, 1514/1642, 1522/1642, 1530/1642, 1538/1642, 1546/1642, 1554/1642, 1562/1642, 1570/1642, 1578/1642, 1586/1642, 1594/1642, 1602/1642, 1610/1642, 1618/1642, 1626/1642, and 1634/1642.
 32. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), and wherein the second antigen-binding domain that specifically binds human PSMA comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3); wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 924, 156, 1484, 1492, 1500, 1508, 1516, 1524, 1532, 1540, 1548, 1556, 1564, 1572, 1580, 1588, 1596, 1604, 1612, 1620, 1628, and 1636; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 926, 158, 1486, 1494, 1502, 1510, 1518, 1526, 1534, 1542, 1550, 1558, 1566, 1574, 1582, 1590, 1598, 1606, 1614, 1622, 1630, and 1638; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:928, 160, 1488, 1496, 1504, 1512, 1520, 1528, 1536, 1544, 1552, 1560, 1568, 1576, 1584, 1592, 1600, 1608, 1616, 1624, 1632, and 1640; A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 932, and 1644; A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 934, and 1646; and A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 936, and 1648; and wherein A2-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124 and 68; A2-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126 and 70; A2-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 128 and 72; A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 932, 164, and 1644; A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 934, 166, and 1646; and A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 936, 168, and
 1648. 33. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises a heavy chain comprising variable domain framework regions having an amino acid sequence selected from FR1 (SEQ ID NO: 1655), FR2 (SEQ ID NO: 1656), FR3 (SEQ ID NO: 1657), and FR4 (SEQ ID NO: 1658).
 34. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain that specifically binds human CD3 comprises a HCVR comprising HCDR1-HCDR2-HCDR3 having the amino acid sequences of SEQ ID NOs: 1659-1660-1661.
 35. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the second antigen-binding domain competes for binding to human PSMA with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124 and 68; A2-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126 and 70; A2-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 128 and 72; A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 932 and 1644; A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 934 and 1646; and A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:168, 936, and
 1648. 36. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the second antigen-binding domain competes for binding to human PSMA with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 122 and 66, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 930 and
 1642. 37. The bispecific antigen-binding molecule of any one of claims 12-24 wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (A1-HCDR1, Al-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 924, 156, 1484, 1492, 1500, 1508, 1516, 1524, 1532, 1540, 1548, 1556, 1564, 1572, 1580, 1588, 1596, 1604, 1612, 1620, 1628, and 1636; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 926, 158, 1486, 1494, 1502, 1510, 1518, 1526, 1534, 1542, 1550, 1558, 1566, 1574, 1582, 1590, 1598, 1606, 1614, 1622, 1630, and 1638; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 928, 160, 1488, 1496, 1504, 1512, 1520, 1528, 1536, 1544, 1552, 1560, 1568, 1576, 1584, 1592, 1600, 1608, 1616, 1624, 1632, and 1640; A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 932, and 1644; A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 934, and 1646; and A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 936, and
 1648. 38. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 922, 154, 1482, 1490, 1498, 1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578, 1586, 1594, 1602, 1610, 1618, 1626, and 1634, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:930, 162, and
 1642. 39. The bispecific antigen-binding molecule of any one of claims 12-24, wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 922, 154, 1482, 1490, 1498, 1506, 1514, 1522, 1530, 1538, 1546, 1554, 1562, 1570, 1578, 1586, 1594, 1602, 1610, 1618, 1626, and 1634, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:930, 162, and 1642; and wherein the second antigen-binding domain competes for binding to human PSMA with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:122 and 66, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 930, 162, and
 1642. 40. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the bispecific antigen-binding molecule of any one of claims 12-39 and a pharmaceutically acceptable carrier or diluent.
 41. A method for treating a cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of claim
 40. 42. The method of claim 41, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer.
 43. The method of claim 42, wherein the cancer is prostate cancer.
 44. The method of claim 43, wherein the prostate cancer is castrate-resistant prostate cancer. 